Phosphatase inhibitor protein-1 as a regulator of cardiac function

ABSTRACT

The present invention relates to novel nucleic acids which encode novel mutant forms of Inhibitor Protein-1 (1-1). In particular, the 1-1 mutant forms comprise altered phosphorylation sites of PKC-α. In addition, the present invention relates to methods of regulating cardiac contractility and function, and for treatment of cardio myopathy and heart failure, which employ the novel nucleic acids and polypeptides. Vectors comprising the novel nucleic acids, Antibodies to the novel proteins, and diagnostic and screening methods associated therewith, are also provided.

RELATED APPLICATIONS/PATENTS & INCORPORATION BY REFERENCE

This application is the U.S. national phase, pursuant to 35 U.S.C. §371,of PCT international application Ser. No. PCT/US2007/003470, filed Feb.9, 2007, designating the United States and published in English on Sep.7, 2007 as publication no. WO 2007/100465 A2, which claims priority toU.S. Provisional Application Ser. No. 60/772,327, filed Feb. 10, 2006.The entire contents of the aforementioned patent applications areincorporated herein by this reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant Nos.DK036569, HL007382, HL026057, HL052318, HL057263, HL057623, HL064018,and HL077101 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 18, 2010, isnamed 65353US5.txt and is 28,307 bytes in size.

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the PCT and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference. More generally, documents orreferences are cited in this text, either in a Reference List before theclaims, or in the text itself; and, each of these documents orreferences (“herein-cited references”), as well as each document orreference cited in each of the herein-cited references (including anymanufacturer's specifications, instructions, etc.), is hereby expresslyincorporated herein by reference.

BACKGROUND OF THE INVENTION

It has been previously established (U.S. Patent App. Pub. No.20050066381) that Protein Kinase C alpha (“PKC-α”) activity is increasedin the pathological state of heart failure. Phosphatase InhibitorProteinI(I-1) is a key regulator of cardiac contractility. I-1 is knownto regulate cardiac contractility by inhibiting the activity of ProteinPhosphatase-1 (“PP-1”). I-1's ability to inhibit PP-1 is further knownto be regulated by phosphorylation. When threonine 35 of I-1 isphosphorylated by Protein Kinase A (PKA), PP-1 activity is inhibited,cardiac contractility is enhanced (Pathak, A., et al. 2005 Circ Res15:756′-66). It was previously shown that serine 67 (S67) is a PKC alphaphosphorylation site, and that a S67 I-1 mutant (for example, S67A) thatmimics a constitutively unphosphorylated state, shows reducedphosphorylation relative to the wild type I-1. However, in vitro testingconditions failed to reveal any inhibition of PP-1 activity.

Heart failure, also called congestive heart failure, is a disorder inwhich the contractility of the heart muscle decreases, and the heartloses its ability to pump blood efficiently. It is estimated to affectover 10 million Americans, alone. Heart failure is almost always achronic, long-term condition, and consumes an inordinate amount ofmedical intervention and human resource dollars. In particular, theconsequences of heart failure to the rest of the body organs can bedevastating both in terms of the overall reduction in productive life ofthe patient, and the expense of treatment. The condition may affect theright side, the left side, or both sides of the heart. As the pumpingaction of the heart is compromised, blood begins backing up into otherareas of the body. Many organs and organ systems begin to suffercumulative damage from lack of oxygen and nutrients.

There may be many underlying causes, and heart failure becomes morecommon with advancing age. Problematically, some patients with heartfailure have no obviously noticeable symptoms, permitting seriousperipheral conditions to manifest without the benefit of earlyintervention to ward off or abate the rate of serious organ damage.Regular screening and early detection will enable a patient to electlife style and dietary changes that will slow progress of the disease.Methods for large-scale screening, and early and accurate detection, aswell as capability to prognose the development of heart failure, beforesignificant organ damage is incurred, are clearly needed. In addition,particularly with elderly patients, there is a need for additionallong-lasting treatment options that do not depend entirely on compliancewith drug product ingestion schedules.

SUMMARY OF THE INVENTION

Accordingly, the instant invention provides novel nucleotide sequenceswhich encode polypeptides comprising novel forms of phophatase inhibitorprotein-1, and functional fragments thereof, that may be employed inmethods of modulating cardiac contractility in animals, includinghumans. The nucleotide sequences may be introduced into cardiac cells,and expression conditions may be triggered, using technology known inthe art. The introduction of genetic material may be for purposes ofincorporation into host genetic material for long-term expressioncapability, or for purposes of shorter, transient expression needs. Inaddition, the expression product itself, in the form of novelpolypeptides comprising novel forms of I-1 may be administered, directlyor indirectly, as the modulating agent, particularly in more acute onsetinstances.

In one aspect, the invention provides an isolated nucleic acid moleculethat encodes a constitutively unphosphorylated phosphatase inhibitor-1protein comprising the amino acid sequence of SEQ ID NO: 5, or aconstitutively unphosphorylated fragment thereof. In one embodiment ofthe invention, the isolated nucleic acid molecule comprises SEQ ID NO:3.

In yet another aspect, the invention provides an isolated nucleic acidmolecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein, wherein the nucleic acid molecule encodes an aminoacid sequence having at least 90% identity (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity) to the amino acid sequence ofSEQ ID NO: 5, or a constitutively unphosphorylated fragment thereof, andwherein the nucleic acid molecule encodes a constitutivelyunphosphorylated amino acid at position 75. In one embodiment of theinvention, the isolated nucleic acid molecule comprises SEQ ID NO: 3 ora nucleotide molecule which is at least 90% identical to the nucleotidesequence of SEQ ID NO: 3.

In another aspect, the invention provides an isolated nucleic acidmolecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein comprising the amino acid sequence of SEQ ID NO: 6,or a constitutively unphosphorylated fragment thereof. In one embodimentof the invention, the isolated nucleic acid molecule comprises SEQ IDNO: 4.

In another aspect, the invention provides an isolated nucleic acidmolecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein, wherein the nucleic acid molecule encodes an aminoacid sequence having at least 90% identity to the amino acid sequence ofSEQ ID NO:6, (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity) or a constitutively unphosphorylated fragment thereof, andwherein the nucleic acid molecule encodes a constitutivelyunphosphorylated amino acid at position 75. In one embodiment of theinvention, the isolated nucleic acid molecule comprises SEQ ID NO: 4 ora nucleotide molecule which is at least 90% identical to the nucleotidesequence of SEQ ID NO: 4.

In yet another aspect, the invention provides an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 5, or a constitutivelyunphosphorylated fragment thereof.

In yet another aspect, the invention provides an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 6, or a constitutivelyunphosphorylated fragment thereof.

In another aspect, the invention provides an isolated nucleic acidmolecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein comprising the amino acid sequence of SEQ ID NO: 12,or a constitutively unphosphorylated fragment thereof. In one embodimentof the invention, the isolated nucleic acid molecule comprises SEQ IDNO: 10.

In yet another aspect, the invention provides an isolated nucleic acidmolecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein, wherein the nucleic acid molecule encodes an aminoacid sequence having at least 90% identity (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity) to the amino acid sequence ofSEQ ID NO: 12, or a constitutively unphosphorylated fragment thereof,and wherein the nucleic acid molecule encodes a constitutivelyunphosphorylated amino acid at position 75. In one embodiment of theinvention, the isolated nucleic acid molecule comprises SEQ ID NO: 10 ora nucleotide molecule which is at least 90% identical to the nucleotidesequence of SEQ ID NO: 10.

In another aspect, the invention provides an isolated nucleic acidmolecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein comprising the amino acid sequence of SEQ ID NO: 16,or a constitutively unphosphorylated fragment thereof. In one embodimentof the invention, the isolated nucleic acid molecule comprises SEQ IDNO: 15.

In yet another aspect, the invention provides an isolated nucleic acidmolecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein, wherein the nucleic acid molecule encodes an aminoacid sequence having at least 90% identity (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity) to the amino acid sequence ofSEQ ID NO: 16, or a constitutively unphosphorylated fragment thereof,and wherein the nucleic acid molecule encodes a constitutivelyunphosphorylated amino acid at position 67 and 75. In one embodiment ofthe invention, the isolated nucleic acid molecule comprises SEQ ID NO:15 or a nucleotide molecule which is at least 90% identical to thenucleotide sequence of SEQ ID NO: 15.

In another aspect, the invention provides an isolated nucleic acidmolecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein comprising the amino acid sequence of SEQ ID NO: 18,or a constitutively unphosphorylated fragment thereof. In one embodimentof the invention, the isolated nucleic acid molecule comprises SEQ IDNO: 17.

In yet another aspect, the invention provides an isolated nucleic acidmolecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein, wherein the nucleic acid molecule encodes an aminoacid sequence having at least 90% identity (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity) to the amino acid sequence ofSEQ ID NO: 18, or a constitutively unphosphorylated fragment thereof,and wherein the nucleic acid molecule encodes a constitutivelyunphosphorylated amino acid at position 67 and 75. In one embodiment ofthe invention, the isolated nucleic acid molecule comprises SEQ ID NO:17 or a nucleotide molecule which is at least 90% identical to thenucleotide sequence of SEQ ID NO: 17.

In another aspect, the invention provides a method of decreasing cardiaccontractility in a subject, the method comprising introducing, intoheart cells of the subject, an effective amount of an isolated nucleicacid molecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein comprising the amino acid sequence of SEQ ID NO: 5or SEQ ID NO: 6, or a constitutively unphosphorylated fragment thereof,thereby decreasing cardiac contractility in the subject.

In another aspect, the invention provides a method of decreasing cardiaccontractility in a subject, the method comprising administering aneffective amount of an isolated polypeptide comprising the amino acidsequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a constitutivelyunphosphorylated fragment thereof, thereby decreasing cardiaccontractility in the subject.

In another aspect, the invention provides a method of treating a subjecthaving heart failure, the method comprising: introducing into heartcells of the subject, a nucleic acid molecule that comprises a sequenceencoding a mutant form of phosphatase inhibitor-1 protein in an amounteffective to decrease phosphatase activity, wherein the mutant formcomprises at least one constitutively unphosphorylated amino acid at aposition that is a PKC-α phosphorylation site in the wild typephosphatase inhibitor-1 protein, thereby treating the subject havingheart failure. In another embodiment, the at least one constitutivelyunphosphorylated amino acid is A (alanine), D (aspartic acid), or C(cysteine) at position 67 or A, D, or C at position 75 in said mutantform of phosphatase inhibitor-1 protein. In yet another embodiment, thenucleic acid molecule has at least 90% identity to a nucleic acidmolecule comprising the sequence selected from the group consisting ofSEQ ID NO: 3, 4, 9, 10, 15, and 17, and wherein the nucleic acidmolecule encodes a constitutively unphosphorylated amino acid atposition 67 or 75. In yet another embodiment, the mutant form ofphosphatase inhibitor-1 protein comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 5. 6, 11, 12, 16, and18. The nucleic acid molecule encoding the mutant form of the proteinmay be selected from the group consisting of SEQ ID NO: 3, 4, 9, 10, 15,and 17. The mutant form of phosphatase inhibitor-1 protein may be a fulllength protein or a constitutively unphosphorylated fragment thereof. Inanother embodiment, the method further comprises obtaining the nucleicacid.

In another embodiment, the method according to the invention furthercomprises introducing a nucleic acid molecule that comprises a sequenceencoding a mutant form of phosphatase inhibitor-1 protein in an amounteffective to decrease phosphatase activity, wherein the mutant formcomprises at least one constitutively phosphorylated amino acid at aposition that is a PKA phosphorylation site in the wild type phosphataseinhibitor-1 protein, thereby treating the subject having heart failure.In yet another aspect, the at least one constitutively phosphorylatedamino acid is D (aspartic acid, GAC, although GAT is likewisecontemplated) or E (glutamic acid, GAG, although GAA is likewisecontemplated) at position 35 in said mutant form of phosphataseinhibitor-1 protein. In yet another embodiment, the nucleic acidmolecule has at least 90% identity to a nucleic acid molecule comprisingSEQ ID NO: 19, and the nucleic acid molecule encodes a constitutivelyphosphorylated amino acid at position 35. In yet another embodiment, themutant form of phosphatase inhibitor-1 protein comprises the amino acidsequence of SEQ ID NO: 20. The nucleic acid molecule encoding the mutantform of the protein may comprise SEQ ID NO: 19. The mutant form ofphosphatase inhibitor protein may be a full length protein or aconstitutively phosphorylated fragment thereof.

In another aspect, the invention provides a method of treating a subjecthaving heart failure, the method comprising: introducing into heartcells of the subject, a nucleic acid molecule encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO: 21 or a fragmentthereof, in an amount effective to decrease phosphatase activity,thereby treating the subject having heart failure. In anotherembodiment, the nucleic acid molecule comprises a sequence encoding apolypeptide comprising at least amino acid positions 1-65 of SEQ ID NO:21, wherein said polypeptide is truncated at a position that is a PKC-αphosphorylation site in SEQ ID NO: 21. The polypeptide may, in yetanother embodiment, be truncated at position 67 or 75 of SEQ ID NO: 21.These truncated forms of I-1 retain their functionality with respect toinhibiting PP-1.

In another embodiment of the invention, the nucleic acid moleculefurther comprises a promoter operably linked to the coding sequence. Inyet another embodiment, the promoter is a constitutive promoter. Instill another embodiment, the promoter is expressed in multiple tissues,wherein one of said tissues is a cardiac muscle tissue. The promoter maycomprise regulatory sequences from any member of the group consistingof: Cytomegalovirus (CMV), cardiac specific troponin T, myosin heavychain, and myosin light chain.

In another embodiment of a method according to the invention, thenucleic acid molecule is introduced by administering a viral deliverysystem comprising a viral particle. In yet another embodiment, the viralparticle comprises a lentiviral particle or an adeno-associated viral(AAV) particle.

In another embodiment of a method according to the invention, thenucleic acid molecule is introduced in an amount effective to result ina condition selected from the group consisting of myocyte shortening,lowering of the time constant for relaxation, and accelerating calciumsignal decay, and combinations thereof. In yet another embodiment, thenucleic acid molecule is introduced in an amount effective to improvethe end-systolic pressure dimension relationship.

In another embodiment of a method according to the invention, thesubject having heart failure has a condition selected from the groupconsisting of ischemia, arrhythmia, myocardial infarction, abnormalheart contractility, and abnormal Ca2+ metabolism, and combinationsthereof, in addition to heart failure. In yet another embodiment, thesubject is human.

In another embodiment of a method according to the invention, flow ofblood through coronary vessels of the heart of the subject isrestricted, and the nucleic acid molecule is introduced into the lumenof a coronary artery in the subject. In yet another embodiment, theheart is pumping while coronary vein outflow is restricted. In yetanother embodiment, flow of blood through the coronary vessels iscompletely restricted. The restricted coronary vessels may comprise,without limitation: the left anterior descending artery (LAD), thedistal circumflex artery (LCX), the great coronary vein (GCV), themiddle cardiac vein (MCV), or the anterior interventricular vein (AIV).In yet another embodiment, the introduction of the nucleic acid moleculeoccurs after ischemic preconditioning of the coronary vessels. In stillanother embodiment, the nucleic acid molecule is injected into the heartof the subject while aortic flow of blood out of the heart isrestricted, thereby allowing the nucleic acid molecule to flow into theheart.

In another embodiment of a method according to the invention, theadministering comprises the steps of: restricting aortic flow of bloodout of the heart, such that blood flow is re-directed to coronaryarteries; injecting the nucleic acid molecule into the lumen of theheart, aorta, or coronary ostia to provide the nucleic acid molecule toa coronary artery; pumping the heart while the aortic flow of blood outof the heart is restricted; and reestablishing the aortic flow of blood.In yet another embodiment, the nucleic acid molecule is injected intothe heart with a catheter. In still another embodiment, the nucleic acidmolecule is directly injected into a muscle of the heart. In stillanother embodiment, the method further comprises evaluating a parameterof heart function in the subject. The parameter of heart function may,without limitation, be one or more of: heart rate, cardiac metabolism,heart contractility, ventricular function, Ca2+ metabolism, andsarcoplasmic reticulum Ca2+ ATPase activity.

In another aspect, the invention provides a method of diagnosing orprognosing heart failure in a subject comprising: obtaining a sample ofcardiac phosphatase inhibitor-1 protein from the subject; and detectingthe presence of at least one phosphorylated PKC-α phosphorylation site,thereby diagnosing or prognosing heart failure in the subject. Inanother embodiment, the at least one phosphorylated PKC-Aphosphorylation site is a T residue at position 75 or a S residue atposition 67 of the cardiac phosphatase inhibitor-1 protein.

In another aspect, the invention provides a recombinant vectorcomprising an isolated nucleic acid molecule that encodes aconstitutively unphosphorylated phosphatase inhibitor-1 proteincomprising the amino acid sequence of SEQ ID NO: 5, or a constitutivelyunphosphorylated fragment thereof. In yet another aspect, the inventionprovides a recombinant vector comprising an isolated nucleic acidmolecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein comprising the amino acid sequence of SEQ ID NO: 6,or a constitutively unphosphorylated fragment thereof.

In another aspect, the invention provides a pharmaceutical compositioncomprising an isolated polypeptide comprising the amino acid sequence ofSEQ ID NO: 5, or a constitutively unphosphorylated fragment thereof anda pharmaceutically acceptable carrier, excipient, or diluent. In yetanother aspect, the invention provides a pharmaceutical compositioncomprising an isolated polypeptide comprising the amino acid sequence ofSEQ ID NO: 6, or a constitutively unphosphorylated fragment thereof anda pharmaceutically acceptable carrier, excipient, or diluent.

In another aspect, the invention provides a pharmaceutical compositioncomprising an isolated nucleic acid molecule that encodes aconstitutively unphosphorylated phosphatase inhibitor-1 proteincomprising the amino acid sequence of SEQ ID NO: 5, or a constitutivelyunphosphorylated fragment thereof, and a pharmaceutically acceptablecarrier, excipient, or diluent. In yet another aspect, the inventionprovides a pharmaceutical composition comprising an isolated nucleicacid molecule that encodes a constitutively unphosphorylated phosphataseinhibitor-1 protein comprising the amino acid sequence of SEQ ID NO: 6or a constitutively unphosphorylated fragment thereof, and apharmaceutically acceptable carrier, excipient, or diluent. In a furtherembodiment, the nucleic acid molecule is present in a viral vectorselected from the group consisting of: recombinant retrovirus,adenovirus, adeno-associated virus, lentivirus, and herpes simplexvirus-1.

In another aspect, the invention provides an antibody raised against anisolated polypeptide comprising the amino acid sequence of SEQ ID NO: 5,or a constitutively unphosphorylated fragment thereof. In anotheraspect, the invention provides an antibody raised against an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 6, or aconstitutively unphosphorylated fragment thereof. In still anotheraspect, the invention provides a diagnostic reagent comprising such anantibody.

In another aspect, the invention provides a kit for treating a subjecthaving heart failure comprising an isolated nucleic acid molecule thatencodes a constitutively unphosphorylated phosphatase inhibitor-1protein comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:6, or a constitutively unphosphorylated fragment thereof, andinstructions for use in accordance with the methods of the invention.The kit can further comprise an isolated nucleic acid molecule thatencodes a mutant form of phosphatase inhibitor-1 protein comprising theamino acid sequence of SEQ ID NO: 20.

In another aspect, the invention provides a kit for treating a subjecthaving heart failure comprising an isolated nucleic acid molecule thatcomprises a sequence encoding a polypeptide comprising the amino acidsequence of SEQ ID NO: 21 or a fragment thereof, and instructions fortreating the subject having heart failure in accordance with the methodsof the invention.

Other aspects of the invention are described in the followingdisclosure, and are within the ambit of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying drawings,incorporated herein by reference. Various preferred features andembodiments of the present invention will now be described by way ofnon-limiting example and with reference to the accompanying drawings inwhich:

FIG. 1. Recombinant I-1 Proteins—(A) Schematic diagram of I-1recombinant proteins. The human I-1 cDNA was cloned into the pGEX-6P3vector for expression as a GST-fusion protein. (B) SEQ ID Nos: 34-38,respectively, in order of appearance, and (C) SEQ ID Nos: 34, 39, 38,and 40, respectively, in order of appearance. (B) and (C) show sequencealignments showing I-1 wild-type in alignment with the recombinantmutants (S67A, T75A, S67A/T75A, S67D, T75D, and S67D/T75D).

FIG. 2. Phosphorylation of Recombinant Human Inhibitor-1 by PKC-α andPKA-Autoradiographs depicting phosphorylation of I-1 by PKC- or by PKA(i.e., radiolabeled phosphoproteins). For the PKC-α assay, the controlsample (C) lacks PKC-A, Ca²⁺ (EGTA present),1,2-diacyl-sn-glycero-3-phospho-L-serine, and phosphatidylserine, butcontains all other components of the assay. For the PKA assay, thecontrol sample (C) lacks PKA and cAMP, but contains all other componentsof the assay. The reactions were initiated by the addition of 0.25 mM[γ³²P] ATP (0.4 mCi/nmol).

FIG. 3. Recombinant adenoviral vectors—Schematic diagram of recombinantadenoviridae expressing I-1. cDNAs were subcloned from the pGEX-6P-3vector into the pSHUTTLE-IRES-hrGFP-1 vector and inserted into theAdEasy-1 viral backbone by homologous recombination.

FIG. 4. Time course of phosphorylation of I-1 and I-1 (S67A) byPKC-α-PKC-α was used to phosphorylate I-1 and I-1 (S67A) in vitro. Atthe indicated times, 20 μl was withdrawn from each mixture, separated on12% SDS-PAGE gels and transferred to nitrocellulose membranes. A)Autoradiograph depicting radiolabeled phosphoproteins. B) The samemembranes were probed with AC1 antibody (1:1000) for detection of totalI-1 and I-1 (S67A) proteins. C) Plot showing the ratio of³²P-incorporated (in both bands of A) per protein (in both bands, whenpresent in B) at different times, quantified by densitometry andcorrected for background. Data represent mean±S.D. of four independentexperiments. In some cases, S.D. is smaller than the symbol size. **,p<0.01; ***, p<0.001.

FIG. 5. Determination of phosphorylation sites—(A) Reverse-phase HPLCshowing separation of the tryptic peptides numbered by HPLC fractions,with peaks 50 and 51 containing the majority of the radioactivityeluting from the column. (B) MALDI-TOF MS spectra showing aphosphorylated peptide with a mass of 1366.90 Da, corresponding to aminoacids 73-82 of the human I-1 sequence (SEQ ID NO: 33). (C) A plot of theradioactivity eluted versus the amino acid position in each cycle ofEdman degradation, showing that the majority of the isotope eluted withthe fourth amino acid. This matches to the peptide ⁷²KKMTRITPTMK⁸² (SEQID NO: 23) detected in the MALDI-TOF data. The line graph showsmean±S.D. (n=three rounds of cpm counted). In most of the cases, S.D. issmaller than the symbol size. (D) Edman degradation detection of theisotope in the third amino acid of the identified sequence (SEQ ID NO:33).

FIG. 6. PKC-α phosphorylation in vitro showed that Ser-67 and Thr-75 arethe primary PKC-α sites on purified human I-1-Purified human I-1, I-1(S67A), I-1 (T75A) and I-1 (S67A/T75A) proteins were phosphorylated byexogenous PKC-A at different 10 times. (A) Autoradiographs depictingradiolabeled phosphoproteins. (B) The same membranes were used to detectI-1 and I-1 mutant proteins by using AC1 antibody (1:1000). (C) Plotshowing amount of ³²P-incorporated into I-1 and I-1 mutant proteins, asquantified by densitometry and corrected for background at thecorresponding phosphorylation time points shown in A. (D) Bar graphdepicting the radioactivity associated with I-1 and its mutants at 45min, as quantified by densitometry and expressed normalized to I-1levels. The bars show mean±S.D. of three independent experiments. **,p<0.01; ***; p<0.001.

FIG. 7. Analysis of the phosphorylation status of purified humaninhibitor-1 by two-dimensional electrophoresis-2-dimensional gelsdepicting migration shifts (induced by PKC-α phosphorylation of I-1) tothe left of protein spots with pi values (from right to left) of 4.9 and4.7. The pI for I-1 dephosphorylated is 5.1 (n=3). Representative partsof 2-D gel images of I-1 samples are shown.

FIG. 8. 2-D gel electrophoresis corroborates that Ser-67 and Thr-75 arethe two primary PKC-A phosphorylation sites on human I-1—Enlargements ofrelevant regions from 2-dimensional gels of I-1 samples are shown. (A)Phosphorylated I-1 wild type appears as three individual protein spotswith pI values of 5.1, 4.9 and 4.7 (from right to left). (B) and (C) Twospots with pIs of 5.1 and 4.9 were observed when Ser-67 or Thr-75 on I-1was substituted by alanine. (D) The simultaneous mutation of Ser-67 andThr-75 abolished any pI migration shift of the protein. A single spotwith a pI of 5.1 appears in the 2-D gel. Dotted circles indicate theexpected location of phosphorylated species in each I-1 mutant incomparison with the wild type. Each 2-D gel was performed 3 times usingpurified proteins from different phosphorylations assays.

FIG. 9. Effect of PKC-α and PKC phosphorylation of I-1 or I-1 mutants onPP1 activity—Plots depicting I-1 inhibitory activity on PP1 monitored inthe presence of: (A) Dephospho-I-1 (filled squares), PKC-α-phospho-I-1(filled triangles), PKA-phospho-I-1 (open squares), andPKC-α+PKA-phospho-I-1 (open triangles); (B) PKC-α-phosphorylated: I-1(S67A) (filled squares), I-1 (T75A) (filled triangles), and I-1(S67A/T75A) (filled circles); and PKA-phosphorylated: I-1 (S67A) (opensquares), I-1 (T75A) (open triangles), and I-1 (S67A/T75A) (opencircles). The activity (nmol/min/ml) associated with each I-1 species isnormalized to the PP1 activity in the absence of I-1 or its mutants.Quantified values represent the average of 7 different experimentsperformed in duplicate (mean±S.D.).

FIG. 10. Two-dimensional Western blot of I-1 in vivo-2-dimensionalWestern blot depicting proteins separated based on pI (isoelectricpoint) and molecular weight. Enlargement of the boxed region from 2-Dgels is shown below the panel.

FIG. 11. Basal Cardiac Myocyte Contractility (T75D mutation)—Bar graphsdepicting change in cardiac contractility (over time) as a function ofthe T75D mutation in I-1. Rate of myocyte contraction is referred to as+dL/dtmax; rate of myocyte relaxation is referred to as −dL/dtmax;contractile force generated is referred to as FS.

FIG. 12. Basal Cardiac Myocyte Contractility (S67D mutation)—Bar graphsdepicting change in cardiac contractility (over time) as a function ofthe S67D mutation in I-1. Rate of myocyte contraction is referred to as+dL/dtmax; rate of myocyte relaxation is referred to as −dL/dtmax;contractile force generated is referred to as FS.

FIG. 13. PKC-α phosphorylation of I-1 at Thr-75 depresses cardiomyocytecontractility—(A) Images of rat cardiac myocytes 24 hrs after infectionat a MOI of 500. Right image shows green fluorescent protein (GFP)expression. (B) I-1 antibody (AC1; 1:1000)-detected overexpression ofthe protein in cardiomyocyte lysates infected with Ad.I-1WT andAd.I-1(T75D). The same membrane was stripped and probed for PP1 (santaCruz, 1:1000). Coomassie-staining of the upper part of the same geldemonstrates equal protein loading and band pattern. (C) Representativetraces of cardiomyocyte mechanics in Ad.GFP (continuous black line),Ad.I-1WT (discontinuous line), and Ad.I-1(T75D) (continuous grey line).Time to 90% relaxation, fractional shortening (FS %), and maximal ratesof contraction and relaxation (dL/dt_(max)) are shown in bar graph form.Total number of cells: 121 (Ad.GFP), 90 (Ad.I-1 WT) and 91(Ad.I-1(T75D)) from 6 hearts. Values represent means±SEM.

FIG. 14. Phosphorylation of I-1 at Ser-67 and/or Thr-75 depressesmyocytes cardiac function—(A) Image of an adult rat cardiomyocyte 24 hrsafter adenoviral infection at a MOI of 500. An antibody specific for I-1(AC1; 1:1000) was used to detect overexpression of the protein inmyocytes infected with: 1) GFP; 2) I-1 WT; 3) I-1 (S67D); 4) I-1 (T75D);and 5) I-1 (S67D/T75D). The upper part of the gel was stained withCoomassie-blue to demonstrate equal protein loading. (B) Fractionalshortening (FS %) and maximal rates of contraction and relaxation(dL/dt_(max), μm/sec) of adenoviral infected-cardiomyocytes are shown inbar graph form.

FIG. 15. Effect of PKA activation on myocytes infected withAd.I-1(S67D), Ad.I-1(T75D) and Ad.I-1(S67D/T75D)—Fractional shortening(FS %) and maximal rates of contraction and relaxation (dL/dt_(max),μm/sec) of infected-cardiomyocytes treated with 0.1 μM forskolin areshown in bar graph form.

FIG. 16. Effect of phosphorylation of I-1 at Thr-75 on the Ca²⁺ affinityof SR Ca²⁺-transport—(A) Plot depicting the initial rates of SRCa²⁺-transport over a wide range of [Ca²⁺] measured for I-1 wild-type,I-1(175D) and GFP proteins expressed in cultured cardiomyocytes. Thedata were normalized to the calculated V_(max) for Ad.I-1WT,Ad.I-1(T75D) and Ad.GFP samples. Curves represent sigmoidal fit obtainedby the OriginLab 5.1 program. Symbols represent the average of threeindividual homogenized myocytes infected with Ad.GFP (solid squares),Ad.I-1WT (empty circles) and Ad.I-1(T75D) (solid circles), assayed perduplicate. (B) Immunoblot depicting total SERCA2a (Affinity Bioreagents,1:1000), total PLN, and calsequestrin (as an internal loading control(Affinity Bioreagents; 1:1000)).

FIG. 17. Effect of phosphorylation of inhibitor-1 at Ser-67 and/orThr-75 on the Ca²⁺ affinity of SR Ca²⁺-transport—plots showing resultsof assessment of the initial rates of SERCA Ca²⁺-transport incardiomyocytes infected with: (A) Ad.GFP; (B) Ad.I-1WT; (C)Ad.I-1(S67D); (D) Ad.I-1(T75D); and (E) Ad.I-1(S67D/T75D). Symbolsrepresent the average of three homogenized myocytes from individualhearts under basal or forskolin treatment, assayed per duplicate. (F)Graph showing the EC₅₀ average values under basal and forskolintreatment for each group. ***, p<0.001 represents comparison of eachgroup vs. GFP, under basal. #, p<0.05; ##, p<0.01, represent comparisonof each group vs. GFP, under forskolin.

FIG. 18. PKC-α phosphorylation of I-1 at Thr-75 enhances PP1activity—(A) Bar graph depicting total phosphatase activity assayed incardiomyocyte lysates (1 μg) infected with Ad.GFP (solid bar), Ad.I-1WT(open bar) or Ad.I-1(T75D) (grey bar). Okadaic acid (10 nM) was added tocell lysates to differentiate type 1 and 2A phosphatase activities.Quantified values represent the average of 4 independent cell lysatesassayed in duplicate and normalized to Ad.GFP (mean±SEM). (B) Bar graphdepicting PP1c (0.5 ng) activity measured for purified recombinant I-1wild-type (solid bar), PKC-α-phosphorylated I-1(S67A) (open bar) andI-1(T75D) (grey bar). Values are normalized to I-1 wild-type. Error barsindicate SEM values for 5 independent experiments, each per duplicate.

FIG. 19. Percentage of inhibition of protein phosphatase-1 activity inadenoviral infected myocytes upon PKA stimulation—Bar graph showingtotal phosphatase activity assayed in forskolin-treated myocyte lysatesoverexpressing: Ad.GFP (black bar); Ad.I-1WT (white bar); Ad.I-1(S67D)(light grey bar); Ad.I-1(T75D) (medium grey bar); and Ad.I-1(S67D/T75D)(dark grey bar). Bars represent the average of 3 independent myocyteslysates assayed per duplicate (mean±SEM).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term “nucleic acid molecule” or “nucleic acidsequence” is intended to refer to polynucleotides that include an openreading frame encoding a polypeptide, and can further include non-codingsequences, such as introns and desirable regulatory sequences (e.g.,promoters, enhancers, transcriptional terminators and the like). Nucleicacid sequences of the invention can encode a specific gene for aselected purpose. The gene can be endogenous to the host cell or can berecombinantly introduced into the host cell, e.g., as a plasmidmaintained episomally or a plasmid (or fragment thereof) that is stablyintegrated into the genome.

As used herein, the term “isolated” means that the referenced materialis removed from the environment in which it is normally found. Thus, anisolated biological material can be free of cellular components, i.e.,components of the cells in which the material is found or produced. Inthe case of nucleic acid molecules, an isolated nucleic acid includes aPCR product, an mRNA band on a gel, a cDNA, or a restriction fragment.Isolated nucleic acids include sequences inserted into plasmids,cosmids, artificial chromosomes, and the like. Thus, a recombinantnucleic acid may constitute an isolated nucleic acid. An isolatedprotein may be associated with other proteins or nucleic acids, or both,with which it associates in the cell, or with cellular membranes if itis a membrane-associated protein. An isolated material may be, but neednot be, purified.

As used herein, the term “complement” of a nucleic acid (nucleotide)sequence refers to a sequence of bases that can form a double-strandedstructure by matching base pairs. The complementary sequence to G-T-A-C,for instance, is C-A-T-G.

As used herein, a “phosphatase inhibitor-1 protein” or “I-1 protein” isa protein, described, for example, by GenBank Accession No.NM_(—)006741, that regulates cardiac contractility by inhibiting theactivity of Protein Phosphatase-1.

In the context of the phosphatase inhibitor-1 protein or I-1 protein,the term “wild-type” refers to the nucleotide sequence of SEQ ID NO: 7encoding Phosphotase Inhibitor Protein-1 (I-1), subunit 1A, and thepolypeptide sequence of SEQ ID NO: 8, and any other nucleotide sequencethat encodes an I-1 protein (having the same functional properties andbinding affinities as the aforementioned polypeptide sequences), such asallelic variants.

Wild-type I-1 includes so-called “functional derivatives” of theprotein. By “functional derivative” is meant a “chemical derivative,”“fragment,” “polymorph” or “variant” of the polypeptide or nucleic acidof the invention. A functional derivative retains at least a portion ofthe function of the protein, which permits its utility in accordancewith the invention. It is well known in the art that, due to thedegeneracy of the genetic code, numerous different nucleic acid sequencecan code for the same amino acid sequence. It is also well known in theart that conservative changes in amino acid can be made to arrive at aprotein or polypeptide that retains the functionality of the original.In both cases, all permutations are intended to be covered by thisdisclosure.

Included within the scope of this invention are the functionalequivalents of the herein-described isolated nucleic acid molecules. Thedegeneracy of the genetic code permits substitution of certain codons byother codons that specify the same amino acid and hence would give riseto the same protein. The nucleic acid sequence can vary substantiallysince, with the exception of methionine and tryptophan, the known aminoacids can be coded for by more than one codon. The encoded amino acidsequence thereof would, however, be preserved.

In addition, the nucleic acid sequence may comprise a nucleotidesequence which results from the addition, deletion or substitution of atleast one nucleotide to the 5′-end and/or the 3′-end, provided that itsaddition, deletion or substitution does not alter the amino acidsequence described herein, which is encoded by the nucleotide sequence.For example, the nucleic acid molecule of the present invention may haverestriction endonuclease recognition sites added to its 5′-end and/or3′-end.

Further, it is possible to delete codons or to substitute one or morecodons with codons other than degenerate codons to produce astructurally modified polypeptide, but one which has substantially thesame utility or activity as the polypeptide produced by the unmodifiednucleic acid molecule. As recognized in the art, the two polypeptidesare functionally equivalent, as are the two nucleic acid molecules thatgive rise to their production, even though the differences between thenucleic acid molecules are not related to the degeneracy of the geneticcode.

A “chemical derivative” of I-1 contains additional chemical moieties notnormally a part of the protein. Covalent modifications of the protein orpeptides may be introduced into the molecule by reacting targeted aminoacid residues of the peptide with an organic derivatizing agent that iscapable of reacting with selected side chains or terminal residues.

The term “fragment” is used to indicate a polypeptide derived from theamino acid sequence of I-1 having a length less than the full-lengthpolypeptide from which it has been derived. Such a fragment may, forexample, be produced by proteolytic cleavage of the full-length protein.Such a fragment may also be obtained recombinantly by appropriatelymodifying the DNA sequence encoding the proteins to delete one or moreamino acids at one or more sites of the C-terminus, N-terminus, and/orwithin the native sequence. Such fragments retain the functional portionof the native I-1.

Another functional derivative intended to be within the scope of thepresent invention is a “variant” polypeptide, which either lacks one ormore amino acids or contains additional or substituted amino acidsrelative to the native polypeptide. Such variants having added,substituted and/or additional amino acids retain the functional portionof the native I-1. A functional derivative of a protein with deleted,inserted and/or substituted amino acid residues may be prepared usingstandard techniques well-known to those of ordinary skill in the art(for example, using site-directed mutagenesis (Adelman et al., 1983, DNA2:183). Alternatively, proteins with amino acid deletions, insertionsand/or substitutions may be conveniently prepared by direct chemicalsynthesis, using methods well-known in the art.

As used herein, the term “mutant” refers to an I-1 polypeptidetranslated from a gene containing a genetic mutation that results in anamino acid sequence that is altered in comparison to the wild-typesequence and results in an altered function of the I-1 polypeptide.

As used herein, the term “phosphatase activity” refers to the activityof phosphatase on the commonly used model protein substrate, MyBP.Herein, Myelin Basic Protein (MyBP) is employed (labeled with ³²P) as asubstrate (binding partner) in measuring change in protein phosphataseactivity.

As used herein, the phrase “constitutively unphosphorylated”, as in a“constitutively unphosphorylated phosphatase inhibitor I-1 protein” or a“constitutively unphosphorylated fragment of phosphatase inhibitor I-1protein” refers to the phosphatase inhibitor-1 protein, or a fragmentthereof, as continuously unphosphorylated in at least one specific aminoacid position under all physiological conditions. In a specificembodiment, the fragment retains at least one of the amino acidpositions 67 or 75 or both, and contains a mutation that removes orreplaces the phosphorylatable hydroxyl groups at that particularresidue. A “constitutively unphosphorylated amino acid of phosphataseinhibitor-1 protein” refers to an amino acid within the polypeptidechain of I-1 protein, or a fragment thereof, that is unphosphorylatedunder all physiological conditions, i.e., through a mutation of theamino acid residue that removes or replaces the phosphorylatablehydroxyl groups at that particular residue.

As used herein, the term “PKC-α phosphorylation site” refers to aspecific amino acid that is phosphorylated by Protein Kinase C, isoformalpha (PKC-α). Like PKA, PKC is a serine/threonine-specific proteinkinase. It phosphorylates serine or threonine residues in its substrate(specifically, it phosphorylates the OH group in the residue).

As used herein, the term “PKA phosphorylation site” refers to a specificamino acid that is phosphorylated by Protein Kinase A (PKA, also knownas cAMP-dependent protein kinase). Each PKA is a holoenzyme thatconsists of two regulatory and two catalytic subunits. Under low levelsof cAMP, the holoenzyme remains intact and is catalytically inactive.When the concentration of cAMP rises (e.g. activation of adenylatecyclases by certain G protein-coupled receptors, inhibition ofphosphodiesterases which degrade cAMP), cAMP binds to the two bindingsites on the regulatory subunits, which then undergo a conformationalchange that releases the catalytic subunits. The free catalytic subunitscan then catalyze the transfer of ATP terminal phosphates to proteinsubstrates at serine or threonine residues.

As used herein, the term “treating” refers to administering an agent inamount, manner, and/or mode effective to improve a condition, symptom,or parameter associated with a disorder or to prevent progression of adisorder, to either a statistically significant degree or to a degreedetectable to one skilled in the art. An effective amount, manner, ormode can vary depending on the subject and may be tailored to thesubject. For example, the mode of administration can include delivery bya virus or virus-like particle. By preventing progression of a disorder,a treatment can prevent deterioration of a disorder in an affected ordiagnosed subject or a subject suspected of having the disorder, butalso a treatment may prevent the onset of the disorder or a symptom ofthe disorder in a subject at risk for the disorder or suspected ofhaving the disorder.

As used herein, the term “heart failure” refers to any disorder in whichthe heart has a defect in its ability to pump adequately to meet thebody's needs. In many cases, heart failure is the result of one or moreabnormalities at the cellular level in the various steps ofexcitation-contraction coupling of the cardiac cells. It is mostfrequently due to a defect in myocardial contraction, which can occurfor many reasons, the most common of which include: ischemic damage tothe myocardium, excessive mechanical resistance to the outflow of bloodfrom the heart, overloading of the cardiac chambers due to defectivevalve function, infection or inflammation of the myocardium, orcongenitally poor myocardial contractile function. (Braunwald, E. 2001Harrison's Principles of Internal Medicine, 15th ed., pp 1318-29).

As used herein, the term “cardiomyopathy” refers to a deterioration offunction of the myocardium (i.e., heart muscle). Cardiomyopathy can beextrinsic (e.g., wherein the primary pathology resides outside of themyocardium itself, for example, caused by ischemia) or intrinsic (e.g.,wherein the weakness in the heart muscle is not due to an identifiableexternal cause).

As used herein, the term “contractility” (as in myocardialcontractility) refers to the performance of cardiac muscle. It is oftendefined as: the intrinsic ability of a cardiac muscle fibre to contractat a given fibre length.

As used herein, the term “end-systolic pressure dimension relationship”(also known as end-systolic pressure-volume relationship) refers to thefollowing linear relationship (Grossman, W., et al. 1977 Circulation56:845-52):P _(ES) =mV _(ES) +b,wherein P_(ES) and V_(ES) are the end-systolic pressure and volume,respectively, m is the slope of the line describing their relations, andb is the pressure at V_(ES)=0. The equation can also be expressed as:P _(ES) =m(V _(ES) −V ₀),wherein V_(o)=−b/m, the volume at P_(ES)=0. End-systolicpressure-dimension relationship is generally considered a powerful indexof ventricular contractility in humans.

As used herein, the term “heart cell” refers to a cell which can be: (a)part of a heart present in a subject, (b) part of a heart which ismaintained ex vivo, (c) part of a heart tissue, or (d) a cell which isisolated from the heart of a subject. For example, the cell can be amuscle cell, such as a cardiac myocyte (cardiomyocyte) or smooth musclecell. Heart cells of the invention can also include endothelial cellswithin the heart, for example, cells of a capillary, artery, or othervessel.

As used herein, the term “heart” refers to the heart organ present in asubject or to a heart organ that is maintained ex vivo, outside asubject.

As used herein, the term “heart tissue” refers to tissue that is derivedfrom the heart of a subject.

As used herein, the term “restricting blood flow” refers tosubstantially blocking the flow of blood through a vessel, e.g., flow ofblood into the distal aorta and its branches. For example, at leastabout 50% of the blood flowing out of the heart is restricted,preferably about 75% and more preferably about 80, 90, or 100% of theblood is restricted from flowing out of the heart. The blood flow can berestricted by obstructing the aorta and the pulmonary artery, e.g., withclamps.

As used herein, the term “obtaining” refers to synthesizing, purchasing,or otherwise acquiring (the nucleic acid or protein).

As used herein, the term “viral delivery system” refers to a viralparticle, e.g., virus or virus like particle that can introduce anucleic acid that includes a non-viral sequence into a mammalian cell.The viral delivery system itself may or may not be competent for viralreplication.

Other definitions appear in context throughout this disclosure.

Additional Embodiments of the Invention

Phosphatase Inhibitor-1 and Mutants Thereof

A fine-tuned regulation of protein kinase and protein phosphataseactivities is essential in the control of the phosphorylation state ofvarious key phosphoprotein substrates, which modulate glycogenmetabolism, protein synthesis, cell division, neuronal signaling andmuscle contraction. A cross-talk between the second messengercAMP-dependent protein kinase (PKA) and the type-I phosphatase (PP1)occurs at the level of an endogenous phosphoprotein, inhibitor-1 (I-1),allowing amplification of the cAMP-signaling cascade. I-1 was firstidentified in rabbit skeletal muscle, but is widely expressed inmammalian tissues and highly conserved across species. This thermostableprotein (Mr 18,700), upon phosphorylation by PKA at Thr-35, becomesactive and potently inhibits PP1, enhancing PKA-mediated proteinphosphorylation. Thr-35 on inhibitor-1 is dephosphorylated byCa^(2i)/calmodulin-dependent protein 2B (PP-2B, calcineurin) and proteinphosphatase 2A (PP-2A), but PP-2B plays a predominant role in thepresence of Ca^(2t). This reversible phosphorylation of inhibitor-1,which is reciprocally regulated by cAMP and calcium, connects theactions of the two major second messengers, resulting in modulation of alarge number of intracellular processes.

In cardiac muscle, the regulation of PP1 by I-1 has been shown to play arole in both basal contractility as well as in the heart's responses toP-adrenergic stimulation. The positive inotropic effect of theP-adrenergic agonist, isoproterenol, is accompanied by 1-1phosphorylation resulting in inhibition of PP1 activity enhances cardiaccontractility by preventing the dephosphorylation of important proteinsinvolved in the contractile state of the heart. Intriguingly, aconstitutively activated form of I-1 (T35D; AA 1-65) not only protectedthe heart from developing hypertrophy induced by pressure-overload, butalso rescued cardiac function in the setting of pre-existing heartfailure, suggesting that I-1 may be a promising candidate in thetreatment of heart failure.

In addition to Thr-35 phosphorylation on I-1, Ser-67 was also found tobe substantially phosphorylated in vitro. It was discovered that theproline-directed kinase, Cdk5, in striatal brain tissue and the neuronalcdc2-like protein kinase, NCLK were both capable of phosphorylatingSer-67 on I-1. Cdk5-mediated phosphorylation had no effect on I-1activity, while NCLK enhanced inhibitory activity. More recently it wasfound that PKC-α, the major isozyme expressed in the mouse and rabbitheart, also phosphorylates Ser-67, and this may reduce the ability ofI-1 to interact with PP1 by 50%, increasing PP1 activity.

Given that both PKC-α and PP1 activities are significantly increased inhuman and experimental heart failure, the present invention is based, inpart, on the discovery that human I-1 is phosphorylated at an additionalsite (Thr-75) by PKC-α. Data presented herein demonstrates that thiskinase phosphorylates Thr-75 to the same extent that it phosphorylatesSer-67; moreover, both residues are phosphorylated independently of eachother. Extensive kinetic analyses indicate that neither of these PKC-αsites inhibits the activity of the catalytic subunit of PP1.Furthermore, neither of these phosphorylated sites interferes with thePKA-mediated inhibitory function of I-1. The discovery of this novelphosphorylation site provides new agents and therapies to treat heartfailure, and, in particular, new approaches to therapy based on theinterplay between increased PP1, PKA, and PKC-α activity underpathophysiological conditions.

Thus, a method of treating a subject having heart failure iscontemplated comprising inhibiting the phosphorylation activity ofPKC-α. Further contemplated is enhancing the phosphorylation activity ofPKA in addition to inhibiting phosphorylation activity of PKC alpha.

A method of treatment according to an embodiment of the presentinvention comprises introduction into the heart cells of the subject, anucleic acid that comprises a sequence encoding a mutant form ofphosphatase inhibitor-1 protein, wherein the mutant form comprises atleast one amino acid at a position that is a PKC-α phosphorylation sitein the wild type, wherein the at least one amino acid is constitutivelyunphosphorylated or mimics an unphosphorylated state in the mutant form.

In a more specific embodiment, the mutant form comprises a mutation thatremoves or replaces the phosphorylatable hydroxyl groups found on theresidue in question (for example, S67 and/or T75). In more specificembodiments, the T75 and/or S67 residue may be substituted or deleted.

For example, the mutant form may comprise at least one amino acid at aposition that is a PKC-α phosphorylation site in the wild type protein,wherein the at least one amino acid is constitutively unphosphorylated.In a specific embodiment, the at least one amino acid is alanine (A),aspartic acid (D), or cysteine (C) at position 65 or alanine (A),aspartic acid (D), or cysteine (C) at position 75 in said mutant form ofphosphatase inhibitor-1 protein. The amino acid substitutions may beselected based on similar charge (no charge), opting for conservativesubstitutions, and based on size (lack of bulk).

In another specific embodiment, the mutant form comprises at least oneamino acid at a position that is a PKA phosphorylation site in the wildtype protein, wherein the at least one amino acid is constitutivelyphosphorylated. For example, the at least one amino acid may be asparticacid (D) or glutamic acid (E) at position 35 in said mutant form ofphosphatase inhibitor-1 protein. Again, the amino acid substitutions maybe selected based on similar charge (negative), as well as on size (lackof bulk).

In embodiments where it is desirable for a residue to mimic thephosphorylated state, such as with the use of a T35 mutant, mutationscomprising the substitution of the residue for glutamic acid or asparticacid are contemplated.

Nucleic Acid Molecules

Nucleic acid molecules of the invention include DNA molecules (e.g.,linear, circular, cDNA or chromosomal DNA) and RNA molecules (e.g.,tRNA, rRNA, mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but advantageously is double-stranded DNA. The nucleicacid molecule of the invention includes a nucleic acid molecule that isfree of sequences that naturally flank the nucleic acid molecule (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid molecule) inthe chromosomal DNA of the organism from which the nucleic acid isderived. Moreover, an isolated nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular materials whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention (for example, a nucleicacid molecule having the nucleotide sequence of SEQ ID NO: 3, 4, 9, 10,15, and 17 can be isolated using standard molecular biology techniquesand the sequence information provided herein. For example, nucleic acidmolecules can be isolated using standard hybridization and cloningtechniques (e.g., as described in Sambrook, J., Fritsh, E. F., andManiatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1989)) or can be isolated by the polymerase chainreaction using synthetic oligonucleotide primers designed based upon thesequence of, for example, SEQ ID NO: 3, 4, 9, 10, 15, and 17. A nucleicacid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. In another embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown, for example, in SEQ ID NO: 3, 4, 9, 10,15, and 17.

The present invention may likewise feature recombinant nucleic acidmolecules (e.g., recombinant DNA molecules) that include nucleic acidmolecules described herein.

Polypeptides

Another aspect of the present invention features polypeptides.

It is well understood that one of skill in the art can mutate (e.g.,substitute) nucleic acids that, due to the degeneracy of the geneticcode, encode for an identical amino acid as that encoded by thenaturally-occurring gene. This may be desirable in order to improve thecodon usage of a nucleic acid to be expressed in a particular organism.Moreover, it is well understood that one of skill in the art can mutate(e.g., substitute) nucleic acids that encode for conservative amino acidsubstitutions. It is further well understood that one of skill in theart can substitute, add or delete amino acids to a certain degreewithout substantially affecting the function of a gene product ascompared with a naturally-occurring gene product, each instance of whichis intended to be included within the scope of the present invention.

In an embodiment, a polypeptide of the present invention has an aminoacid sequence shown in SEQ ID NO: 5, 6, 11, 12, 16, and 18.

Sequence Identity

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows. Todetermine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, 60%, and even more preferably at least 70%,80%, 90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused unless otherwise specified) are a Blossum 62 scoring matrix with agap penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller ((1989)CABIOS, 4:11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

Gene Transfer/Delivery

Introduction is contemplated to be via any technology either known orcurrently unknown, that achieves the result of the desired introduction.The nucleic acids described herein can be incorporated into a geneconstruct to be used as a part of a gene therapy protocol. Methods forgene transfer in vivo are known in the art. Approaches include insertionof the subject gene in viral vectors including recombinant retroviruses,adenovirus (e.g., replication deficient, first generation, or gutted,second generation, adenovirus), adeno-associated virus (e.g., the viralcapsid may be an AAV capsid such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, or AAV11 capsid; one skilled in the art wouldknow that there are, likely, other variants not yet identified thatperform the same or similar function; or may include components from twoor more AAV capsids, as described in U.S. Pat. No. 6,491,907),lentivirus, and herpes simplex virus-1, or recombinant bacterial oreukaryotic plasmids. To produce a lentiviral particle and other viralparticles, the nucleic acid that encodes the agent of interest isoperably linked to a packaging signal. The nucleic acid is packaged incells that express viral structural proteins. For example, the cells caninclude nucleic acids that encode the viral structural proteins, butthat lack a packaging signal.

Adeno-associated virus is a nonpathogenic human parvovirus, capable ofsite-specific integration into chromosome 19 (Fisher et al., NatureMedicine (1997). Replication of the virus, however, requires a helpervirus, such as an adenovirus (Fisher et al., Nature Medicine (1997). AnAAV coding region can be replaced with nonviral genes, and the modifiedvirus can be used to infect both dividing and non-dividing cells (Xiaoet al., J. Virol. (1996); Kaplitt et al., Ann. Thorac. Surg. (1996)).Exemplary methods for the preparation and use of AAVs are described inFisher et al., Nature Medicine (1997) Xiao et al., J. Virol. (1996),Kaplitt et al., Ann. Thorac. Surg. (1996).

Different recombinant AAV genome structures are described in WO01/092551, including duplexed parvovirus vectors—a parvovirus particlecomprising a parvovirus capsid (e.g., an AAV capsid) and a vector genomeencoding a heterologous nucleotide sequence, where the vector genome isself-complementary, i.e., the vector genome is a dimeric invertedrepeat.

Viral vectors transfect cells directly; plasmid DNA can be deliveredwith the help of, for example, cationic liposomes (lipofectin) orderivatized (e.g. antibody conjugated), polylysine conjugates,gramacidin S, artificial viral envelopes or other such intracellularcarriers, as well as direct injection of the gene construct orCaPO.sub.4 precipitation carried out in vivo.

Gene transfer into cardiovascular tissue, for example, has beensuccessful using adenovirus (Ad) vectors with strong, non-tissuespecific gene expression cassettes driven by cytomegalovirus (CMV) orRous sarcoma virus (RSV) promoters. Clinical trials involvingtransduction of cardiac cells with viral vectors to deliver angiogenicfactors such as vascular endothelial cell growth factor (VEGF),fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) havebeen ongoing. Intra-aorta or intracoronary injection of virus has beenused in vivo in animal models. As is known from studies on cysticfibrosis, transduction of all cells in a tissue is not required forimproved function.

Tissue specific promoters have been used to increase specificity ofmyocardial gene expression (Rothmann, et al., Gene Ther. (1996)).Another strategy to restrict expression of transferred genes to theheart has involved direct injection of a viral vector into themyocardium (Gutzman, et al, Cric. Res. (1993); French, et al., (1994),Circulation. (1994)). Another attempt involved intrapericardial virusvector injection combined with proteinase treatment (Fromes, et al.,Gene Ther. (1999)). These manipulations achieved local gene delivery,although with some drawbacks, due to a lack of intense viral vectordiffusion.

The efficiency of cardiomyocyte gene delivery by an adeno-associatedvirus (AAV) vector was documented in vitro using cultured rat neonatalcells, as well as in an ex vivo system using rat papillary muscleimmersion (Maeda, et al., J. Mol. Cell. Cardiol. (1998)). Ex vivo AAVvector transfer followed by syngeneic heart transplantation was reportedto achieve high efficiency marker gene expression (Svensson et al.,Circulation. (1999)). Methods of achieving a high level of in vivocardiotopic gene transfer with high consistency (average 60-70% ofcardiac myocytes) are described, e.g., in US Published Application20020032167. Other methods for the preparation and use of viral vectorsare described in WO 96/13597, WO 96/33281, WO 97/15679, and Trapnell, etal., Curr. Opin. Biotechnol. (1994); Ardehali, et al., J. Thorac.Cardiovasc. Surg. (1995); Dalesandro, et al., J. Thorac. Cardiovasc.Surg. (1996); Sawa, et al., Circ (1995); Lee, et al., J. Thorac.Cardiovasc. Surg. (1996); Yap, et al., Circ. (1996); and Pellegrini, etal., Transpl. Int. (1998).

A subject polynucleotide can also be administered using a non-viraldelivery vehicle. “Non-viral delivery vehicle” (also referred to hereinas “non-viral vector”) as used herein is meant to include chemicalformulations containing naked or condensed polynucleotides (e.g., aformulation of polynucleotides and cationic compounds (e.g., dextransulfate)), and naked or condensed polynucleotides mixed with an adjuvantsuch as a viral particle (i.e., the polynucleotide of interest is notcontained within the viral particle, but the transforming formulation iscomposed of both naked polynucleotides and viral particles (e.g.,adenovirus particles) (see, e.g., Curiel, et al. Am. J. Respir. CellMol. Biol. (1992)). Thus “non-viral delivery vehicle” can includevectors composed of polynucleotides plus viral particles where the viralparticles do not contain the polynucleotide of interest.

“Non-viral delivery vehicles” include bacterial plasmids, viral genomesor portions thereof, wherein the polynucleotide to be delivered is notencapsidated or contained within a viral particle, and constructscomprising portions of viral genomes and portions of bacterial plasmidsand/or bacteriophages. The term also encompasses natural and syntheticpolymers and co-polymers. The term further encompasses lipid-basedvehicles. Lipid-based vehicles include cationic liposomes such asdisclosed by Felgner, et al (U.S. Pat. Nos. 5,264,618 and 5,459,127;PNAS 84:7413-7417, (1987); Annals N.Y. Acad. Sci. (1995); they may alsoconsist of neutral or negatively charged phospholipids or mixturesthereof including artificial viral envelopes as disclosed by Schreier,et al. (U.S. Pat. Nos. 5,252,348 and 5,766,625).

Non-viral delivery vehicles include polymer-based carriers.Polymer-based carriers may include natural and synthetic polymers andco-polymers. Preferably, the polymers are biodegradable, or can bereadily eliminated from the subject. Naturally occurring polymersinclude polypeptides and polysaccharides. Synthetic polymers include,but are not limited to, polylysines, and polyethyleneimines (PEI;Boussif, et al., PNAS 92:7297-7301, (1995)), which molecules can alsoserve as condensing agents. These carriers may be dissolved, dispersedor suspended in a dispersion liquid such as water, ethanol, salinesolutions and mixtures thereof. A wide variety of synthetic polymers areknown in the art and can be used.

A preparation that includes units of a viral delivery system can bedelivered to heart cells of a subject (in vivo or ex vivo) by any of avariety of methods known in the art.

In clinical settings, the gene delivery systems for the therapeutic genecan be introduced into a patient by any of a number of methods, each ofwhich is familiar in the art. For instance, a pharmaceutical preparationof the gene delivery system can be introduced systemically, e.g. byintravenous injection, and specific transduction of the protein in thetarget cells occurs predominantly from specificity of transfectionprovided by the gene delivery vehicle, cell-type or tissue-typeexpression due to the transcriptional regulatory sequences controllingexpression of the receptor gene, or a combination thereof. In otherembodiments, initial delivery of the recombinant gene is more limitedwith introduction into the animal being quite localized. For example,the gene delivery vehicle can be introduced by catheter (see U.S. Pat.No. 5,328,470) or by stereotactic injection (e.g. Chen, et al. PNAS 91:3054-3057 (1994)).

Administration routes include intravenous, intradermal, subcutaneous,oral (e.g., inhalation or ingestion), transdermal (topical), andtransmucosal. Also contemplated is injection, e.g., intra-arterially,intramuscularly, intra-pericardially, or intravenously.

In one exemplary implementation, the preparation is directly injectedinto heart tissue. U.S. Ser. No. 10/914,829 describes a protocol fordirect injection. Direct injection or application of a viral vector intothe myocardium can restrict expression of the transferred genes to theheart (Gutzman et al, Cric. Res. (1993); French et al., Circulation.(1994)). The preparation may also be provided to cells ex vivo. Cellscontaining the protein of interest (e.g., mutant I-1) are thenadministered to the patient.

In another exemplary implementation, the preparation is introduced intothe lumen of one or more coronary arteries. Passage of blood out of thecoronary arteries can be restricted. The preparation can be deliveredantegrade and allowed to reside in the arteries for between one to fiveminutes, e.g., between one to three minutes.

In another exemplary implementation, the preparation is affixed tosupport matrices (e.g., sutures, surgically implanted materials, grafts,and the like) to provide controlled or uncontrolled release into thelocal tissue and/or vascular environment, as described in WO 01/091803.

Non-viral vehicles may be delivered by similar methods.

Pharmaceutical Compositions

An isolated nucleic acid molecule or polypeptide according to theinvention can be incorporated into pharmaceutical compositions suitablefor administration to a subject, e.g., a human. Such compositionstypically include the polypeptide or nucleic acid molecule and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances are known. Except insofaras any conventional media or agent is incompatible with the activecompound, such media can be used in the compositions of the invention.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition can be formulated to be compatible with itsintended route of administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propylene glycoland liquid polyethylene glycol, and the like), and suitable mixturesthereof.

The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the optimal particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be achieved by various antibacterialand anti-fungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., an isolated nucleic acid molecule as described herein)in the optimal amount in an appropriate solvent with one or acombination of ingredients enumerated above, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thetherapeutic agent into a sterile vehicle which contains a basicdispersion medium and other ingredients from those enumerated above. Inthe case of sterile powders for the preparation of sterile injectablesolutions, preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art.

The therapeutic agent can be prepared with a carrier(s) that willprotect it against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The pharmaceutical preparation of the gene therapy construct can alsocomprise a slow release matrix in which the gene delivery vehicle isimbedded. Recombinant parvoviruses, and in particular, recombinantadeno-associated virus (rAAV), for example, can be used to delivernucleic acid sequences (i.e., genes and DNA sequences) for gene therapy(as described above) following a dehydration or drying step (i.e.,partial or complete desiccation, lyophilization), in which thetherapeutic virus vector is dried onto (i.e., affixed to) a supportmatrix. Useful support matrices include surgically implantable materials(i.e., sutures, surgical graft material, implantable devices and thelike) for packaging and transport to a subject, thus allowing deliveryof gene therapy via the rAAV affixed to the support matrixes. This isdescribed further in WO 01/091803. Alternatively, where the completegene delivery system can be produced intact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can comprise one ormore cells which produce the gene delivery system.

The nucleic acid molecule to be delivered can also be formulated as aDNA- or RNA-liposome complex formulation. Such complexes comprise amixture of lipids that bind to genetic material (DNA or RNA) by means ofcationic charge (electrostatic interaction). Cationic liposomes whichmay be used in the present invention include3.beta.-[N—(N′,N′-dimethyl-aminoethane)-1-carbamoyl]-cholesterol(DC-Chol), 1,2-bis(oleoyloxy-3-trimethylammonio-p-ropane (DOTAP) (see,for example, WO 98/07408), lysinylphosphatidylethanol-a-mine (L-PE),lipopolyamines such as lipospermine,N-(2-hydroxyethyl)-N,N-d-dimethyl-2,3-bis(dodecyloxy) 1-propanaminiumbromide, dim ethyl dioctadecyl ammonium bromide (DDAB),dioleoylphosphatidyl ethanolamine (DOPE), dioleoylphosphatidyl choline(DOPC), N(1,2,3-dioleyloxy) propyl-N,N,N-triethylammonium (DOTMA),DOSPA, DMRIE, GL-67, GL-89, Lipofectin, and Lipofectamine (Thiery, etal. Gene Ther. (1997); Felgner, et al., Annals N.Y. Acad. Sci. (1995);Eastman, et al., Hum. Gene Ther. (1997)). Polynucleotide/lipidformulations described in U.S. Pat. No. 5,858,784 can also be used inthe methods described herein. Many of these lipids are commerciallyavailable from, for example, Boehringer-Mannheim, and Avanti PolarLipids (Birmingham, Ala.). Also encompassed are the cationicphospholipids found in U.S. Pat. Nos. 5,264,618, 5,223,263 and5,459,127. Other suitable phospholipids that may be used includephosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingomyelin, phosphatidylinositol, and the like. Cholesterol may alsobe included.

Administration

A pharmaceutical composition as described above can be injected into anaffected vessel, e.g., an artery, or an organ, e.g., the heart. In onemethod of treatment embodiment, flow of blood through coronary vesselsof a heart is restricted and a viral delivery system is introduced intothe lumen of a coronary artery. In a specific embodiment, the heart ispermitted to pump while coronary vein outflow is restricted. In anotherspecific embodiment, the viral delivery system is injected into theheart while restricting aortic flow of blood out of the heart, therebyallowing the viral delivery system to flow in to and be delivered to theheart. In other embodiments, the flow of blood through the coronaryvessels is completely restricted, and in specific such embodiments, therestricted coronary vessels comprise: the left anterior descendingartery (LAD, the distal circumflex artery (LCX), the great coronary vein(GCV), the middle cardiac vein (MCV), or the anterior interventricularvein (AIV). In certain embodiments, the introduction of the viraldelivery system occurs after ischemic preconditioning of the coronaryvessels.

In another embodiment, the viral delivery system comprising a vector isinjected into the heart by a method comprising the steps of: restrictingaortic flow of blood out of the heart, such that blood flow isre-directed to coronary arteries; injecting the vector into lumen of theheart, aorta or coronary ostia such that the vector flows into thecoronary arteries; permitting the heart to pump while the aortic flow ofblood out of the heart is restricted; and reestablishing the aortic flowof blood. In a more specific embodiment, the vector is injected into theheart with a catheter, and in an even more specific embodiment, thevector is directly injected into a muscle of the heart.

PKC-α inhibition constitutes a pharmacological target for treatment ofheart failure, given that PKC-α activity is increased in thepathological state of heart failure. Hence, the administration of PKC-αantagonists or any agent which acts to inhibit PKC-α activity incombination with the nucleic acids or the polypeptides of the presentinvention. In conditions where it may be desirable to decrease cardiaccontractility, administration of pharmacological agents which act asPKC-α agonists is additionally indicated.

Evaluation of Treatment

A treatment method of the invention can be evaluated by assessing theeffect of the treatment on a parameter related to cardiac function orcardiac cellular function, e.g., without limitation, heart rate, cardiacmetabolism, heart contractility, ventricular function, Ca2+ metabolism,and sarcoplasmic reticulum Ca2+ ATPase activity.

A treatment can also be evaluated by its effect on a subject, e.g.,according to parameters that one skilled in the art of treatment wouldrecognize as relevant for the particular treatment. For example, intreating heart failure, exemplary parameters may relate to cardiacand/or pulmonary function. Cardiac parameters include pulse, EKGsignals, lumen loss, heart rate, heart contractility, ventricularfunction, e.g., left ventricular end-diastolic pressure (LVEDP), leftventricular systolic pressure (LVSP), Ca²⁺ metabolism, e.g.,intracellular Ca²⁺ concentration or peak or resting Ca²⁺, forcegeneration, relaxation and pressure of the heart, a force frequencyrelationship, cardiocyte survival or apoptosis or ion channel activity,e.g., sodium calcium exchange, sodium channel activity, calcium channelactivity, sodium potassium ATPase pump activity, activity of myosinheavy chain, troponin I, troponin C, troponin T, tropomyosin, actin,myosin light chain kinase, myosin light chain 1, myosin light chain 2 ormyosin light chain 3, IGF-1 receptor, P13 kinase, AKT kinase,sodium-calcium exchanger, calcium channel (L and T), calsequestrin orcalreticulin. The evaluation can include performing angiography (e.g.,quantitative angiography) and/or intravascular ultrasound (IVUS), e.g.,before, after, or during the treatment.

Methods of Diagnosing/Prognosing Heart Failure

Additionally contemplated herein is diagnosing or prognosing heartfailure in a subject by obtaining a sample of cardiac phosphataseinhibitor-1 protein from the subject and detecting the presence of atleast one phosphorylated PKC alpha phosphorylation site, morespecifically, wherein at least one phosphorylated PKC alphaphosphorylation site comprises T75 or S67. The diagnostic reagent maycomprise the inventive isolated nucleic acid, or a complement orfragment thereof.

Kits

The isolated nucleic acid molecule or polypeptide of the invention canbe provided in a kit. The kit may include, without limitation, (a) thenucleic acid molecule or polypeptide, e.g., a composition that includesthe nucleic acid molecule or polypeptide, and (b) informationalmaterial. The informational material can be descriptive, instructional,marketing or other material that relates to the methods described hereinand/or the use of the nucleic acid molecule or polypeptide of theinvention for the methods described herein. For example, theinformational material may relates to heart failure.

In one embodiment, the informational material can include instructionsto administer the nucleic acid molecule or polypeptide of the inventionin a suitable manner to perform the methods described herein, e.g., in asuitable dose, dosage form, or mode of administration (e.g., a dose,dosage form, or mode of administration described herein). In anotherembodiment, the informational material can include instructions toadminister the nucleic acid molecule or polypeptide of the invention toa suitable subject, e.g., a human, e.g., a human having, or at risk for,heart failure. For example, the material can include instructions toadminister the nucleic acid molecule or polypeptide of the invention toa subject who has or is at risk for having cardiomyopathy.

In addition to the isolated nucleic acid molecule or polypeptide of theinvention, the composition of the kit can include other ingredients,such as a solvent or buffer, a stabilizer, a preservative, and/or asecond agent for treating heart failure. Alternatively, the otheringredients can be included in the kit, but in different compositions orcontainers than the nucleic acid molecule or polypeptide of theinvention. In such embodiments, the kit can include instructions foradmixing the nucleic acid molecule or polypeptide of the invention andthe other ingredients, or for using the nucleic acid molecule orpolypeptide of the invention together with the other ingredients.

The nucleic acid molecule or polypeptide of the invention can beprovided in any form, e.g., liquid, dried or lyophilized form. It ispreferred that the nucleic acid molecule or polypeptide of the inventionbe substantially pure and/or sterile. When the nucleic acid molecule orpolypeptide of the invention is provided in a liquid solution, theliquid solution preferably is an aqueous solution, with a sterileaqueous solution being preferred. When the nucleic acid molecule orpolypeptide of the invention is provided as a dried form, reconstitutiongenerally is by the addition of a suitable solvent. The solvent, e.g.,sterile water or buffer, can optionally be provided in the kit.

The kit can include one or more containers for the compositioncontaining the nucleic acid molecule or polypeptide of the invention. Insome embodiments, the kit contains separate containers, dividers orcompartments for the composition and informational material. Forexample, the composition can be contained in a bottle, vial, or syringe,and the informational material can be contained in a plastic sleeve orpacket. In other embodiments, the separate elements of the kit arecontained within a single, undivided container. For example, thecomposition is contained in a bottle, vial or syringe that has attachedthereto the informational material in the form of a label. In someembodiments, the kit includes a plurality (e.g., a pack) of individualcontainers, each containing one or more unit dosage forms (e.g., adosage form described herein) of the agent. For example, the kitincludes a plurality of syringes, ampules, foil packets, or blisterpacks, each containing a single unit dose of the nucleic acid moleculeor polypeptide of the invention. The containers of the kits can be airtight and/or waterproof. The kit optionally includes a device suitablefor administration of the composition, e.g., a stent, syringe, or anyuseful delivery device.

Antibodies

Antibodies that selectively bind an isolated polypeptide comprising theamino acid sequence of SEQ ID NO: 5 or 6, or a constitutivelyunphosphorylated fragment thereof, are likewise contemplated. Methods ofpreparing antibodies are well known to those of ordinary skill in thescience of immunology. As used herein, the term “antibody” means notonly intact antibody molecules, but also fragments of antibody moleculesthat retain immunogen-binding ability. Such fragments are also wellknown in the art and are regularly employed both in vitro and in vivo.Accordingly, as used herein, the term “antibody” means not only intactimmunoglobulin molecules but also the well-known active fragmentsF(ab′)₂, and Fab. F(ab′)₂, and Fab fragments that lack the Fc fragmentof intact antibody, clear more rapidly from the circulation, and mayhave less non-specific tissue binding of an intact antibody (Wahl etal., J. Nucl. Med. 24:316-325 (1983). The antibodies of the inventioncomprise whole native antibodies, bispecific antibodies; chimericantibodies; Fab, Fab′, single chain V region fragments (scFv), fusionpolypeptides, and unconventional antibodies.

In one embodiment, an antibody that binds an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 5 or 6, or aconstitutively unphosphorylated fragment thereof, is monoclonal.Alternatively, the antibody is a polyclonal antibody. The preparationand use of polyclonal antibodies are also known the skilled artisan. Theinvention also encompasses hybrid antibodies, in which one pair of heavyand light chains is obtained from a first antibody, while the other pairof heavy and light chains is obtained from a different second antibody.Such hybrids may also be formed using humanized heavy and light chains.Such antibodies are often referred to as “chimeric” antibodies.

In general, intact antibodies are said to contain “Fc” and “Fab”regions. The Fc regions are involved in complement activation and arenot involved in antigen binding. An antibody from which the Fc′ regionhas been enzymatically cleaved, or which has been produced without theFc′ region, designated an “F(ab′)₂” fragment, retains both of theantigen binding sites of the intact antibody. Similarly, an antibodyfrom which the Fc region has been enzymatically cleaved, or which hasbeen produced without the Fc region, designated an “Fab′” fragment,retains one of the antigen binding sites of the intact antibody. Fab′fragments consist of a covalently bound antibody light chain and aportion of the antibody heavy chain, denoted “Fd.” The Fd fragments arethe major determinants of antibody specificity (a single Fd fragment maybe associated with up to ten different light chains without alteringantibody specificity). Isolated Fd fragments retain the ability tospecifically bind to immunogenic epitopes.

Antibodies can be made by any of the methods known in the art utilizingan isolated polypeptide comprising the amino acid sequence of SEQ ID NO:5 or 6, or a constitutively unphosphorylated fragment thereof, orimmunogenic fragments thereof, as an immunogen. One method of obtainingantibodies is to immunize suitable host animals with an immunogen and tofollow standard procedures for polyclonal or monoclonal antibodyproduction. The immunogen will facilitate presentation of the immunogenon the cell surface. Immunization of a suitable host can be carried outin a number of ways. Nucleic acid sequences encoding an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 5 or 6, ora constitutively unphosphorylated fragment thereof, or immunogenicfragments thereof, can be provided to the host in a delivery vehiclethat is taken up by immune cells of the host. The cells will, in turn,express the receptor on the cell surface generating an immunogenicresponse in the host. Alternatively, nucleic acid sequences encoding anisolated polypeptide comprising the amino acid sequence of SEQ ID NO: 5or 6, or a constitutively unphosphorylated fragment thereof, orimmunogenic fragments thereof, can be expressed in cells in vitro,followed by isolation of the receptor and administration of the receptorto a suitable host in which antibodies are raised.

Alternatively, antibodies against an isolated polypeptide comprising theamino acid sequence of SEQ ID NO: 5 or 6, or a constitutivelyunphosphorylated fragment thereof, may, if desired, be derived from anantibody phage display library. A bacteriophage is capable of infectingand reproducing within bacteria, which can be engineered, when combinedwith human antibody genes, to display human antibody proteins. Phagedisplay is the process by which the phage is made to ‘display’ the humanantibody proteins on its surface. Genes from the human antibody genelibraries are inserted into a population of phage. Each phage carriesthe genes for a different antibody and thus displays a differentantibody on its surface.

Antibodies made by any method known in the art can then be purified fromthe host. Antibody purification methods may include salt precipitation(for example, with ammonium sulfate), ion exchange chromatography (forexample, on a cationic or anionic exchange column preferably run atneutral pH and eluted with step gradients of increasing ionic strength),gel filtration chromatography (including gel filtration HPLC), andchromatography on affinity resins such as protein A, protein G,hydroxyapatite, and anti-immunoglobulin.

Antibodies can be conveniently produced from hybridoma cells engineeredto express the antibody. Methods of making hybridomas are well known inthe art. The hybridoma cells can be cultured in a suitable medium, andspent medium can be used as an antibody source. Polynucleotides encodingthe antibody of interest can in turn be obtained from the hybridoma thatproduces the antibody, and then the antibody may be producedsynthetically or recombinantly from these DNA sequences. For theproduction of large amounts of antibody, it is generally more convenientto obtain an ascites fluid. The method of raising ascites generallycomprises injecting hybridoma cells into an immunologically naivehistocompatible or immunotolerant mammal, especially a mouse. The mammalmay be primed for ascites production by prior administration of asuitable composition (e.g., Pristane).

Monoclonal antibodies (Mabs) produced by methods of the invention can be“humanized” by methods known in the art. “Humanized” antibodies areantibodies in which at least part of the sequence has been altered fromits initial form to render it more like human immunoglobulins.Techniques to humanize antibodies are particularly useful when non-humananimal (e.g., murine) antibodies are generated. Examples of methods forhumanizing a murine antibody are provided in U.S. Pat. Nos. 4,816,567,5,530,101, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.

The present invention may be more fully understood by reference to thefollowing supporting experiments and examples. However, it is understoodthat the examples are intended to elucidate certain aspects of thepresent invention, and should not be construed as limiting the scope ofthe invention as defined by the claims.

EXAMPLES

The present inventors undertook examination of PKC-α mediatedphosphorylation of I-1, using purified proteins. cDNAs, encoding humanI-1 or an I-1 mutant with alanine substitution at Ser-67 were cloned andexpressed. The obtained recombinant proteins were purified and theGST-tag was removed. PKC-α phosphorylation of the pure proteinsindicated that ³²P-incorporation into the mutant is decreased but notcompletely abolished in comparison to the I-1 wild type, suggesting thatthere may be another PKC-α phosphorylation site. For identification ofthis putative PKC-α site, phosphorylated human I-1 was subjected tomatrix-assisted laser desorption ionization mass spectrometry incombination with Edman degradation. These analyses revealed threonine-75as a new PKC-α site on human I-1. To confirm this data, I-1 mutants withalanine substitutions at Thr-75 (T75A), and Ser-67 plus Thr-75(S67A/T75A) were generated.

PKC-α treatment of I-1 and its mutants showed reduced ³²P-incorporationinto either S67A or T75A and none in the S67A/T75A mutant. Furtheranalysis by two-dimensional electrophoresis corroborated that: 1) Thr-75is a PKC-α site; and 2) Ser-67 and Thr-75 are the only residuesphosphorylated by PKC-α on human I-1. To determine the functionalsignificance of Thr-75 phosphorylation, protein phosphatase assays wereperformed. Phosphorylation of I-1 or I-1 mutants by PKA was associatedwith inhibition of PP1. However, PKC-α phosphorylation of I-1 had noeffect on its activity. Furthermore, PKC-α phosphorylation had no effecton the PKA-mediated inhibitory function of I-1.

Materials

PKC-α, PKA and cAMP were purchased from Upstate Biotechnology.Phosphatidylserine was obtained from Avanti Polar-Lipids. The pGEX 6P-3plasmid, Gluthathione Sepharose 4B, PreScission Protease and ImmobilineDryStrips, IPG Buffer pH 4-7 were obtained from Amersham Biosciences.Quick-Change II site-directed mutagenesis kits and BL21 CodonPlus(DE3)-RIPL Competent Cells were obtained from Stratagene.Diacylglycerol, ampicillin and IPTG were obtained from Sigma-Aldrich.SYPRO Ruby Protein Gel Stain was obtained from Cambrex. T4 ligase, EcoRIand Not I restriction enzymes were purchased from New England Biolabs.Protein Desalting Spin Columns and B-PER GST Fusion Protein PurificationKit were purchased from Pierce. [γ-32P] ATP was obtained from PerkinElmer. Anti-AC1 was a custom-made (Affinity Bioreagents) rabbitpolyclonal affinity-purified antibody against the N-terminal sequence ofmouse I-1 (¹MEPDNSPRKIQFTVP¹⁵) (SEQ ID NO: 24). Anti-GST rabbitpolyclonal antibody was obtained from Affinity Bioreagents.

Methods

Generation of Inhibitor-1 Mutant Proteins

The human I-1 cDNA (GenBank Accession #U48707) was cloned into thepGEX-6P-3 vector in-frame with and on the C-terminal side of theGlutathione-S-Transferase (GST) gene (FIG. 1A). The forward cloningprimer was: 5′-CAGA GAATTC C ATG GAG CAA GAC AAC AGC CC-3′ (SEQ ID NO:25) (EcoRI restriction enzyme site underlined; spacer nucleotide forin-frame expression shaded; start codon italicized), and the reversecloning primer was: 5′-CAGA GCGGCCGC TCA GAC CGA GTT GGC TCC CT -3′ (SEQID NO: 26) (Not I restriction enzyme site underlined; stop codonitalicized). The PreScission Protease cleavage site was located betweenthe GST and I-1 genes to facilitate subsequent removal of the GST tag.Mutations of the I-1 cDNA were obtained in the pGEX-6P3 vector; usingthe QuikChange II Site-Directed Mutagenesis Kit.

The primers used for mutagenesis of Ser-67 to Ala were: 5′-TCC ACT TTGGCA ATG GCA CCA CGG CAA CGG AAG AA-3′ (SEQ ID NO: 27) (alanine codonunderlined) and its complement (FIG. 1B). For mutagenesis of Thr-75 toAla, the primers were: 5′-CGG CAA AAG AAG ATG GCA AGG ATC ACA CCC AC-3′(SEQ ID NO: 28) (alanine codon underlined) and its complement. I-1(S67A/T75A) was generated by using I-1 (S67A) as a template formutagenesis of Thr-75 to Ala; the same set of primers described abovefor I-1 (T75A) was used (FIG. 1B). The primers used for mutagenesis ofSer-67 to Asp were: 5′-TCC ACT TTG GCA ATG GAC CCA CGG CAA CGG AAG AA-3′(SEQ ID NO: 29) (asparatate codon underlined) and its complement (FIG.1C). For mutagenesis of Thr-75 to Asp, the primers were: 5′-CGG CAA CGGAAG AA ATG GAC AGG ATC ACA CCC AC-3′ SEQ ID NO: 30) (aspartate codonunderlined) and its complement (FIG. 1C) I-1 (S67D/T75D) was generatedin a stepwise manner, as described above (FIG. 1C).

Each of these plasmids was transfected into BL21 CodonPlus (DE3)-RIPLcompetent cells and grown on the LB-agar ampicillin (150 μg/ml) plates.Individual colonies were inoculated into 3-ml LB-ampicillin (50 μg/ml)starter cultures and grown at 37° C. for 16 hours. 1 ml of thesecultures was inoculated into 100 ml of LB-ampicillin and grown at 25° C.for 2 hours. At this point, sterile IPTG was added to a finalconcentration of 0.1 uM and the cultures were grown for an additional 4hours at 25° C. The cells were then pelleted, and the GST-I-1 fusionproteins were purified using the B-PER GST Fusion Protein PurificationKit. GST fusion proteins were extensively dialyzed against 50 mMTris-HCl (pH 7.0) and incubated with PreScission Protease for 4 hours at4° C. After proteolytic cleavage, the PreScission enzyme and GST tagwere removed from the medium, using pre-washed Glutathione Sepharose 4Bfor 4 hours or overnight at 4° C. Samples were analyzed by SDS-PAGEusing 15% polyacrylamide gels as described by Laemmli (24) to estimatethe extent of cleavage and protein yield after purification. Proteinconcentration was determined by Micro BC assay (Pierce).

In Vitro Phosphorylation Assays

Reactions were conducted at 35° C. in 150 μl of buffer containing 7 μgof I-1 or I-1 mutant proteins. Recombinant I-1 or I-1 (S67A), I-1(T75A), I-1 (S67A/T75A) mutants were phosphorylated by PKC-α. For PKC-α(3 μg/ml) phosphorylation, the final concentrations were 50 mM Tris-HCl(pH 7.0), 5 mM MgCl₂, 5 mM NaF, 0.5 mM CaCl₂, 0.3 mM phosphatidylserineand 0.02 mM 1,2-Diacyl-sn-glycero-3-phospho-L-serine. Thephosphorylation reactions were initiated by the addition of 0.25 mM[γ-³²P] ATP (0.4 mCi/nmol). At indicated times, 20 ul was withdrawn fromeach mixture and the reactions were stopped by adding 4 ul of SDS samplebuffer (5-strength) to the medium. For two-dimensional electrophoresis,25 μg (35 μg in some cases) of protein were phosphorylated by PKC-α (4ug/ml) in 100 μl of buffer at 35° C. for 45 minutes (or overnight insome cases), as described above. Reactions were initiated by theaddition of 400 μM ATP. In all cases, Ca²⁺ (1 mM EGTA present),Phosphatidylserine, diacylglycerol and PKC-α were omitted from themixture for control samples.

Recombinant I-1 or I-1 (S67D/T75D) mutant (7 μg) were phosphorylated byPKA. PKA (0.1 μg) phosphorylation was performed in the presence of 50 mMTris-HCl (pH 7.0), 5 mM MgCl₂, 5 mM NaF, 1 mM EGTA, 1 μM cAMP and 0.25mM [γ-³²P] ATP (0.4 mCi/nmol) or 400 μM ATP. After 1 hour, the reactionswere stopped by adding SDS sample buffer to the medium. For the controlsamples, PKA and cAMP were omitted from the reaction medium. The amountof [³²P]-phosphate into I-1 species was determined via SDS-PAGE andautoradiography or by trichloroacetic acid (20%, w/v) precipitationfollowed by dialysis. Densitometric analysis of the data was conductedusing ImageQuant 5.2 software.

In initial experiments, PKC-A phosphorylated I-1 was observed to migrateas a doublet of phosphoproteins on autoradiographs. Further studiesrevealed that this occurred only upon PKC-α but not PKA phosphorylation(FIG. 2). This doublet was still observed when each of the two PKC-Aphosphorylation sites was mutated to Ala (FIGS. 4 and 6). Interestingly,the I-1 doublet was related to the presence of the PKC-α activator,1,2-diacyl-sn-glycero-3-phospho-L-serine (DAG), in the phosphorylationbuffer (data not shown). This fact may be due to the alteration of theprotein's net charge and, thus, reducing the binding of SDS.

Identification of Additional Phosphorylation Sites on Inhibitor-1

To identify novel PKC-α phosphorylation sites, 10 ug of GST-I-1(purified I-1) were incubated in 50 μl of PKC-α phosphorylation bufferas described above in the presence of trace [γ-³²P] ATP for 4 h at 37°C. The reaction mixture was subjected to 12% SDS-PAGE and the gel wasstained with SYPO Ruby overnight at room temperature. The ³²P-labeledGST-I-1 band was identified, excised from the gel and subjected totrypsin digestion. Tryptic peptides were separated using a Vydac C18reverse-phase HPLC column, and the fractions were immediately essayedfor ³²P by Cerenkov counting. Radioactive peaks were subjected tomatrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF)mass spectrometry and Edman degradation. The GPMAW program was used tomatch experimental peptide masses against the predicted peptides derivedfrom the I-1 human sequence.

Immunoblot Analysis

I-1 species were separated by SDS-PAGE using 12% polyacrylamide gels.Following separation, proteins were transferred to nitrocellulosemembranes (pore size 0.1 um) (Schleicher & Schuell Bioscience) by wettransfer (180 mA for 3 h). Nonspecific binding sites were blocked for1-2 h at room temperature, using 5% dried milk in Tris-buffered saline(pH 7.4) containing 0.1% Tween 20. Membranes were probed for 3 h at roomtemperature or overnight at 4° C. with primary antibodies ACI (1:1000)for I-1 or anti-GST (1:1000). A secondary peroxidase-labelled antibody(Amersham Biosciences) was used in combination with an enhancedchemiluminescent detection system (Supersignal West PicoChemiluminescent, Pierce) to visualize the primary antibodies. Theoptical density of the bands was analyzed by ImageQuant 5.2 software.

Two-Dimensional Electrophoresis

Purified I-1 or PKC-α- or PKA-phosphorylated I-1 and mutants were (oncedesalted using Protein Desalting Spin Columns) solubilized inrehydration buffer consisting of 7 M Urea, 2 M Thiourea, 4% CHAPS, 10 mMDTT, 1% IPG 4-7 Buffer and 0.01% bromophenol blue. The solubilizedproteins were applied to 18 cm Immobiline™ Drystrips (pH 4-7NL) andincubated overnight at room temperature. The rehydration isoelectricfocusing (IEF) was carried out for 60,000 volt-hours at 50 mA per stripon a Genomic Solutions Investigator 2-D Electrophoresis System. Thesecond dimension was run on 12.5% slab gels for 14 hours at 500 V. Thegels were fixed and stained with a fluorescent stain (SYRPO Ruby)overnight at room temperature. SYPRO stained gels were scanned using anFLA-3000 Imager (Fuji Medical Systems, Stamford, Conn.) with 475 nmfluorescent laser and a yellow 520 nm filter. For comparison purposes,2-D gels were processed in parallel and subsequently, ProImage softwarewas used to localize the spots to a standard set of coordinates.Therefore, gels can be compared to each other, and changes in themigration pattern of protein spots upon experimental manipulation can beeasily detected.

Trichloroacetic acid precipitated protein samples from mouse cardiachomogenates were solubilized in DeSteak Solubilization buffer andsubsequently applied to 11 cm IPG strips (pH 3-10) for isoelectricfocusing. After protein separation in the second dimension usingSDS-PAGE 12.5% proteins were transferred to nitrocellulose membranes(pore size 0.1 um) by wet transfer (180 mA for 3 h). The same proceduredescribed above for immunoblot analysis of 1-D gels was applied here.

Isolation of Inhibitor-I

I-1 was isolated from mouse cardiac muscle according to the proceduredescribed by Shenolikar et al. (25) with modifications. Briefly, frozentissues (1.5 g) was pulverized with a mortar in liquid nitrogen andhomogenized with a Polytron homogenizer in 2 ml of ice-coldphosphate-buffered saline (pH 7.2). Immediately, 5 ml of 1.5% (w/v)trichloroacetic acid were added and the homogenate was rotated for 1 hat 4° C., before subjecting to centrifugation at 9,000 rpm for 30 min.The supernatant was adjusted to 15% (w/v) trichloroacetic acid, rotatedat 4° C. overnight, and centrifuged at 18,000 rpm for 30 min. The pelletwas resuspended in 0.5 M Tri-HCl, pH 8.0 ( 1/20 of the original extractvolume), boiled for 10 min and centrifuged as above. The pH of thesupernatant was adjusted to −7, using 1M NaOH, and the sample wassubjected to 2-D gel electrophoresis.

Protein Phosphatase Activity Assays

Assays for protein phosphatase activity were performed in a 50 μlreaction mixture containing 50 mM Tris-HCl (pH 7.0), 0.1 mM Na₂EDTA, 5mM DTT, 0.01% Brij 35, and, optionally, 0.5 ng of PP1 (New EnglandBioLabs). The reaction was initiated by adding 10 ul of a standardsubstrate ³²P-labeled Myelin Basic Protein (MyBP) (final concentrationof 50 μM). To generate the ³²P-MyBP substrate, commercially purifiedMyBP was previously phosphorylated by PKA to a stoichiometry of 2-4 molphosphate per mol, following manufacturer's instructions (New EnglandBioLabs) and stored at 4° C. After 10 min at 30° C., the reaction wasterminated by adding 200 ul of 20% trichloroacetic acid, cooled on iceand centrifuged. The amount released [³²P] in the assay was determinedby scintillation counting 200 ul from the supernatant. A blank reactionin which PP1 was omitted from the mixture was carried out in parallel.

To measure the I-1 inhibitory activity on PP1, I-1 wild type and itsmutants (0.1 mg/ml) were phosphorylated prior to addition to the PP1activity assay by PKC-α or PKA for the 1 h or overnight at 30° C. in thepresence of 400 uM ATP as described above, omitting NaF from the assaymedium. Dual phosphorylation by both kinases, PKA and PKC-α, was carriedout stepwise as follows. After PKC-α incubation, an aliquot waswithdrawn from the mixture and EGTA (a mM), cAMP (1 uM), PKA (0.1 ug)and ATP (400 uM) were added to the medium. The PKA phosphorylationreaction was incubated at 30° C. for the same time employed for PKC-αtreatment. Subsequently, dephosphorylation of [³²P] MyBP by PP1 wasmonitored in the presence of dephosphorylated or phosphorylated I-1 orseveral I-1 mutants.

Generation of the I-1 Adenoviridae

To generate an ex vivo expression vector for assessment of thefunctional effects of phosphorylation of I-1 at a specific site, the I-1cDNA bearing the specific mutation (for example, Thr-75 to Asp mutation(T75D)) was first cloned into the pShuttle-IRES-hrGFP-1 vector. Thisvector was allowed to homologously recombine with the AdEasy-1adenovirus backbone vector (FIG. 3). Thus, the Ad-Easy XL system wasused to generate adenoviruses encoding I-1 wild-type (Ad. I-1WT), I-1mutants I-1(S67D), I-1(T75D), I-1 (S67D/T75D), or green fluorescentprotein (Ad.GFP) in Ad-293 cells. This resulted in areplication-deficient recombinant adenovirus, which can express both I-1(for example, the T75D mutant) and green fluorescent protein (GFP). Theadenoviridae were amplified, purified (using the Adenovirus MiniPurification Kit (Virapur)), and titered (using the Adeno-X Rapid TiterKit (Clontech)) according to standard procedures.

Adenovirus-Mediated Gene Transfer and Myocyte Contractility

To characterize the effects of phosphorylation of Inhibitor-1 atspecific sites on cardiac contractility, ventricular myocytes from adultmale Sprague-Dawley rats (≈300 grams) were isolated by collagenasedigestion as previously detailed (Fan et al. Circ. Res. (2004)). Ratswere handled as approved by the Institutional Animal Care and UseCommittee at the University of Cincinnati. Myocytes were resuspended inmodified culture medium (M199, Gibco), counted, plated on laminin-coatedglass coverslips or dishes, and infected with adenoviruses at amultiplicity of infection of 500 for 2 h, at 37° C. in a humidified 5%CO₂ incubator. Myocyte contraction at basal level and, optionally, underForskolin (100 nM) (Sigma-Aldrich) treatment, was performed by using aGrass S5 stimulator (0.5 Hz, square waves) 24 h after infection.Fractional shortening (FS %), time to 90% relaxation (% of baseline) andmaximal rates of contraction and relaxation (dL/dT_(max)) werecalculated using a video edge motion detector (Crescent Electronics).For immunoblotting, cultured cardiomyocytes were harvested and lysed for30 minutes at 4° C. in lysis buffer as described previously (Fan et al.Circ. Res. (2004)).

For immunoblotting, cultured cardiomyocytes were harvested andhomogenized using a Polytron in solubilization buffer containing 50 mMTris-HCl (pH 7.0), 10 mM NaF, 1 mM EDTA, 0.3 mM sucrose, 0.3 mM PMS F,0.5 mM DL-Dithiothreitol (DT) and protease inhibitor cocktail (1 ml per20 gram of tissue) (Sigma). For measurement of protein phosphataseactivity, NaF was omitted from the buffer.

Sarcoplasmic Reticulum Ca²⁺ Uptake in Cultured Rat Cardiomyocytes

After 24 h infection of isolated rat cardiomyocytes, cells were washedtwice with PBS, harvested and homogenized at 4° C. in 50 mM potassiumphosphate buffer (pH=7.0), 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, 0.3 mMPMSF and 0.5 mM DTT. Initial SR Ca²⁺ uptake rates were determined inhomogenates using the Millipore filtration technique and ⁴⁵CaCl₂, aspreviously described (Kiss et al. Circ. Res. (1995)). Briefly, 100-250μg of homogenate were incubated at 37° C. in reaction buffer containing40 mM Imidazole (pH=7.0), 95 mM KCl, 5 mM NaN₃, 5 mM MgCl₂, 0.5 mM EGTA,and 5 mM K₂C₂O₄. The initial uptake rates were determined over a widerange of calcium values (pCa 5 to 8). Calcium uptake into cardiomyocyteswas initiated by addition of 5 mM ATP, and aliquots were filteredthrough a 0.45 μm Millipore filter after 0, 30, 60 and 90 seconds toterminate the reaction. The specific ⁴⁵Ca²⁺ uptake values (maximum Ca²⁺uptake rate, V_(max), and concentration of half-maximal Ca-uptake, EC₅₀)were analyzed using the OriginLab 5.1 program.

Autoradiography and Data Analysis

As mentioned briefly, above, the amount of ³²P incorporation into theI-1 species was determined by autoradiography. After wet transfer,nitrocellulose membranes were exposed to Blue Lite Autorad Films(IscBioExpress) for 24 or 48 h, and densitometric analysis of the datawas conducted using Image Quant 5.2 software.

Statistics

All the values are expressed as mean±SEM for n experiments. Comparisonswere evaluated by Student's t-Test for unpaired data or one-way ANOVA,as appropriate. *, p<0.05; **, p<0.01; ***, p<0.001.

Example 1 Phosphorylation of I-1 and I-1 Mutants by PKC-α

As noted, previous work showed that PKC-α phosphorylates inhibitor-1(I-1) at Ser-67. To examine whether additional PKC-α phosphorylationsites may exist on I-1, an I-1 mutant in which Ser-67 was substitutedwith alanine was phosphorylated in vitro by PKC-α as described above.FIG. 4A shows that although PKC-α phosphorylation of the mutant I-1(S67A) is greatly decreased in comparison to wild type I-1, it is notcompletely abolished. Densitometric analysis of ³²P-incorporated perprotein revealed that at steady-sate (20-45 min), I-1 (S67A)incorporated 40±8.6% of the radioactivity levels present in wild types(100%) (FIG. 4C). In some of the experiments, a single additional bandappeared at 78 Kda, which correspond to PKC-α autophosphorylation. Noother radioactive bands were detected in any of the experiments. Aspecific antibody (AC1, 1:1000) recognized the phospho-bands asinhibitor-1 (FIG. 4B). Thus, these results indicate that at least oneadditional PKC-α phosphorylation site exists on 1-1.

Example 2 Phosphorylation Site Determination

To determine the location of the additional PKC-α site (s) on I-1,recombinant purified I-1 was purified and subjected to in vitrophosphorylation by PKC-α in the presence of [γ-³²P] ATP. The ³²P-labeledI-1 was purified by SDS-PAGE, and digested with trypsin. Afterpurification of the tryptic peptides by reverse-phase HPLC, two peaks,50 and 51 (FIG. 5A), contained 62% of the radioactivity eluting from theVydac column. Both fractions were subjected to MALDI-TOF massspectrometry analysis. Mass matching analysis from fraction 51 yieldedthree potential phosphorylated peptides. The detected masses (predictedmass plus phosphate group) were 1226.44, 1494.45, and 1572.68 Da, whichcorresponded to each of the following I-1 sequences: ⁶²STLAMSPRQR⁷¹ (SEQID NO: 31), ⁷²KKMTRITPTMK⁸² (SEQ ID NO: 23), and ¹³⁵KTAECIPKTHER¹⁴⁶ (SEQID NO: 32) (data not shown). In parallel, analysis of peak 50 detected apeptide of mass 1366.46 Da, which corresponded to the sequence⁷³KMTRITPTMK⁸² (SEQ ID NO: 33) (same sequence found in peak 51 minus thefirst lysine) (data not shown).

For the purpose of identifying the amino acid position of the phosphategroup, Edman degradation was performed on fraction 51, since itcontained more radioactivity compared to peak 50. The fifteen cycles ofradio-sequencing carried out on this fraction, showed that the majorityof the radioactive signal eluted with the fourth amino acid(approximately 370 cpm above background) (FIG. 5C). Subsequent readingof this result against the MALDI mass matches, showed that only thepeptide ⁷²KKMTRITPTMK⁸² (SEQ ID NO: 23) possessed a phosphorylatableamino acid at position four (FIGS. 5B and 5C; phosphoamino ishighlighted in a shaded box). A small amount of isotope eluted with thethird amino acid (120 cpm), but this was likely due to an alternativetrypsin cleavage between lysine 72 and 73 of I-1 (⁷³KMTRITPTMK⁸² (SEQ IDNO: 33)). The amount of isotope detected in position 5 was due to thecarryover of the previous site, a common artifact of this technique(usually about 50%). After cycle 4, the cpm decreased from cycle tocycle by roughly the same percentage. The peptide, corresponding to thesequence ⁷³KMTRITPTMK⁸² (SEQ ID NO: 33), was found to be phosphorylatedat its third amino acid, Thr-75 (FIG. 5D). Thus, threonine-75 (Thr-75)was identified as a novel PKC-x phosphorylation site on human I-1.

To further confirm the identity of Thr-75 as a phosphorylation site forPKC-α, and to determine whether Ser-67 and Thr-75 are the only PKC-αsites of human I-1, alanine substitution mutations at Thr-75 [I-1(T75A)] and at Ser-67 plus Thr-75 [I-1 (S67A/T75A)] were made asdescribed above. As shown in FIG. 6 (A and C), the I-1 (T75A) mutantincorporated significantly less ³²P upon PKC-α incubation in comparisonto I-1 wild type. ACI antibody recognized all these phospho-bands asinhibitor-1 (FIG. 6B). Densitometric analysis of ³²P-incorporated perprotein at 45 min reveals that I-1 (T75A) incorporated 33.8±12.7% of thelevels in wild type (100%) (FIG. 6D). I-1 wild-type was phosphorylatedby PKC-α for 45 min to a stoichiometry of 0.88 mol Pi/mol protein,whereas the incorporation of Pi into I-1(S67A) and I-1(75A) were reducedto 0.35 and 0.30 mol Pi/mol protein, respectively. FIGS. 6A, 6C, and 6Dshow incorporation of ³²P into I-1 wild-type and mutants in in vitrotime course reactions, expressed as densitometric units.

The results indicate that Thr-75 is, indeed, a PKC-α site on human I-1.Although the mutation of Thr-75 to Ala was associated with a greaterdecrease in ³²P-incorporation, compared to the I-1 (S67A) mutant, thisdifference was not statistically significant (FIG. 6D). Therefore, PKC-αappears to phosphorylate human I-1 in vitro at Thr-75 to the same degreethat it phosphorylates Ser-67. Moreover, mutation of both Ser-67 andThr-75 to Ala abolishes ³²P-incorporation into I-1 (FIGS. 6A, 6C, and6D). These data indicate that, under the conditions described herein,these are two primary PKC-α phosphorylation sites on purified human I-1protein.

Example 3 Analysis of PKC-α Phosphorylation of I-1 by Two-DimensionalElectrophoresis

To further corroborate the autoradiography results, 2-D gelelectrophoresis analysis was used to detect possible mobility changes inI-1 due to PKC-α phosphorylation. 2-D gel electrophoresis separatesproteins based on both their isoelectric point (pI) and molecularweight. Phosphorylation causes the pI value of a protein to become moreacidic, but has a negligible effect on its molecular weight. Analysis ofthe non-phosphorylated I-1 gel image indicated that the protein migratesin a 2-D gel as a single spot at a pI of 5.1 and molecular weight of ˜30kD (FIG. 7A). In the PKC-α phosphorylated sample, three spots werevisible, with pIs of (from right to left): 5.1, 4.9 and 4.7,corresponding to non-phosphorylated, singly phosphorylated, and doublyphosphorylated protein, respectively (FIG. 7B). A higher concentrationof phosphorylated I-1 (35 ug) subjected to 2D gel electrophoresis didnot show any additional phosphorylation shifts (data not shown).Attempts to increase the degree of inhibitor-1 phosphorylation byincreasing the concentration of PKC-α, ATP or duration of the incubationtime, did not reveal any additional phosphoprotein spots either. Noother proteins spots were detected in the samples.

Since the studies above indicated that Thr-75 is a new phosphorylationsite for PKC-α, purified human I-1 wild type, I-1 (S67A), I-1 (M75A) andI-1 (S67A/T75A) proteins were incubated with PKC-α and subjected inparallel to 2-D gel electrophoresis. When either Ser-67 or Thr-75 weremutated to Ala, only two spots with pI values of 5.1 and 4.9 weredetected in the gels (FIGS. 8B and 8C). These data demonstrated thatblocking one of the two PKC-α sites prevented the incorporation ofphosphate into the protein, resulting in a two-spot pattern in contrastto the three-spot observed in phosphorylated I-1 wild-type (FIG. 8A).Moreover, simultaneous mutation of Ser-67 and Thr-75 completelyabolished any migration shift of I-1 to the left (FIG. 8D), furtherestablishing that: 1) Thr-75 is a PKC-α site; and 2) Ser-67 and Thr-75are the primary PKC-α sites on human I-1.

Example 4 Effects of PKC-α Phosphorylation on I-1 Function

Previous studies have reported that I-1 inhibits PP1 only uponphosphorylation by PKA (6-8). Based on the present finding that PKC-αindependently phosphorylates I-1 to the same extent on two sites, Ser-67and Thr-75, the effect of PKC-α phosphorylation on the function of I-1was examined. In these studies, I-1 wild type untreated or I-1 wild typephosphorylated by: PKC-α, PKA or PKC-α+PKA was used in proteinphosphatase assays (FIG. 9A). Neither dephospho-I-1 norPKC-α-phospho-I-1 inhibited PP1 activity at any of the concentrationstested (0-15 nM). However, PKA-phospho-I-1 inhibited PP1 with an IC₅₀value of 3.2±0.08 nM, consistent with previous reports (7-9).

To investigate whether PKC-α phosphorylation may affect the inhibitoryfunction of PKA-phospho-I-1, I-1 wild type was first phosphorylated byPKC-α and then by PKA as described above. As shown in FIG. 9A,pre-phosphorylation by PKC-α had no effect on the PKA-mediatedinhibitory function of I-1 (IC₅₀ value of 4.9±0.74 nM). Furthermore,none of the following mutants: I-1(S76A), I-1 (T75A) or I-1 (S67-A/T75A)had any inhibitory effect on PP1 function following theirphosphorylation by PKC-α (FIG. 9B).

To further corroborate these results, aspartate substitution mutations,which mimic phosphorylation of I-1 (9, 12), were made at Thr-75 [1-1(T75D)] and at Ser-67 plus Thr-75 [I-1 (S67D/T75D)] as described inabove. None of these mutations had any inhibitory effect on PP1activity. Thus, although PKC-A can phosphorylate I-1 at two distinctsites, these phosphorylations do not inhibit PP1 activity. However, uponPKA phosphorylation, all of these I-1 species were capable of fullyinhibiting PP1 activity (FIG. 9B).

Example 5 Analysis of the Phosphorylation Status of Inhibitor-1 fromMouse Cardiac Tissue

It was previously reported that inhibitor-1 is phosphorylated in vivo atonly two positions, Thr-35 and Ser-67. In an effort to investigate ifThr-75 is also phosphorylated in vivo, a fraction enriched in I-1 wasisolated from mouse cardiac muscle (1.5 g), as described above. Thefinal pellet (˜0.25% of the initial protein) was subjected to 2-D gelelectrophoresis. After the second dimension of electrophoresis, theproteins were electroblotted onto a nitrocellulose membrane andincubated with the antibody AC1 (1:1000) for I-1. As shown in FIG. 10,four protein spots with pI values of 5.1, 4.9, 4.7 and 4.5 (from rightto left) were identified. The streak detected on the right part of themembrane was due to incomplete isoelectric focusing of the protein, andit was observed due to the increased sensitivity of immunoblotting.

Analysis of this image, using ProImage software, indicated that the spotwith a pI of 5.1 corresponded to dephosphorylated recombinant I-1. Theseresults demonstrated that 1-1 is post-translationally modified threetimes in vivo under basal conditions. Given that Thr-35 and Ser-67 areknown to be phosphorylated in vivo, and that the isoelectric pointshifts of I-1 are equivalent to those observed using recombinantprotein, the four spots likely represent dephosphorylated I-1 and I-1phosphorylated at one, two and three sites (Thr-35, Ser-67 and Thr-75).

Example 6 I-1 Mutations and Cardiac Contractility

To test the effect of I-1 mutations on cardiac contractility, adult ratcardiomyocytes were infected with recombinant adenovirus expressingeither wild-type 1-1, 1-1 or T75D under the control of the CMV promoter.Empty vector was used as a control. The rates of myocyte contraction andmyocyte lengthening were determined after stimulation by a Grass S5stimulator (0.5 Hz, square waves). Expression of I-1 T75D significantlyreduced the rates of myocyte contraction (+dL/dtmax decreased by 29%)and myocyte relaxation (−dL/dtmax decreased by 33%) (FIG. 11).Furthermore, the total contractile force generated by myocytesexpressing the mutant protein was reduced 35% or more. The dataindicates that phosphorylation at T75 inhibits contractile functioningin the heart.

To test the effect of the S67D mutation on cardiac contractility, adultrat cardiomyocytes were infected with recombinant adenovirus expressingeither wild-type I-1, or I-1 S67D under the control of the CMV promoter.Empty vector was used as a control. The rates of myocyte contraction andmyocyte lengthening were determined after stimulation by a Grass S5stimulator (0.5 Hz, square waves). Expression of I-1 S67D significantlyreduced the rates of myocyte contraction (+dL/dtmax decreased by 24%)and myocyte relaxation (−dL/dtmax decreased by 28%) (FIG. 12).Furthermore, the total contractile force generated by myocytesexpressing this mutant protein was reduced 35% or more (38%, FIG. 12).This data indicates that phosphorylation at S67 negatively impactscontractile function in the heart.

Next, adult rat cardiomyocytes were infected with either the I-1wild-type adenovirus, Ad.I-1WT, or the constitutively phosphorylated I-1at Thr-75, Ad.I-1(T75D). An adenovirus expressing only GFP, Ad.GFP, wasused as control. Infection efficiency reached nearly 100% after 24 h, asassessed by green fluorescence (FIG. 13A). Ad.I-1 WT and Ad.I-1 (175D)showed the expected overexpression of I-1, whereas endogenous I-1 wasundetectable in cells infected with Ad.GFP (FIG. 13B).

Constitutive phosphorylation of I-1 at Thr-75 induced a markedattenuation in the maximum rates of myocyte shortening (dL/dt_(max);31%) and relengthening (dL/dt_(max); 36%), as well as in the fractionalshortening (33%), compared with either I-1-infected wild-type orGFP-infected cells (FIG. 13C). Time to 90% relaxation was significantlyincreased (by 22%) in Ad.I-1(T75D) infected myocytes. These findingsindicate that phosphorylation of 1-1 at Thr-75 significantly depressesmyocyte contractility.

Next, adult rat cardiomyocytes were infected with Ad.GFP, Ad.I-1WT,Ad.I-1(S67D), Ad.I-1(T75D) and Ad.I-1(S67D/T75D). Adenoviraltransfection efficiency was assessed after 24 hours by greenfluorescence and Western blot immunodetection. As shown in FIG. 14A, thelevels of I-1 expression were similar in all the groups, whereasendogenous I-1 was undetectable in cells infected with Ad.GFP, similarto previous observations (Rodriguez et al. J. Biol. Chem. (2006),El-Armouche et al. Cardiovasc. Res. (2001)). Consistent with recentfindings (Rodriguez et al. J. Biol. Chem. (2006)), expression of theAd.I-1 (T75D) in cultured myocytes significantly reduced the rates ofcontraction and relaxation (29% and 35.5%, respectively), as well asfractional shortening (290%), compared to myocytes expressing Ad.I-1WT(FIG. 14B). 15-20 myocytes/heart were analyzed with a total number ofhearts per group of: 12 (Ad.GFP), 6 (Ad.I-1 WT), 5 (Ad.I-1(S67D)), 8(Ad.I-1(T75D)), and 5 (Ad.I-1(S67D/T75D)).

Expression of the Ad.I-1(S67D) induced similar decreases in the rates ofmyocyte contraction (22.1%) and relaxation (27%), as well as fractionalshortening (25.3%) (FIG. 14B). Although the functional performance ofmyocytes expressing Ad.I-1 (T75D) tended to be more attenuated thanthose expressing Ad.I-1 (S67D), the values were not differentstatistically. Interestingly, expression of the constitutively dualphosphorylated I-1 at S67D and T75D, Ad.I-1(S67D/T75D), yielded similarresults to those elicited by each of the single mutants. Ad.I-1(S67D/T75D) reduced the maximal velocities of contraction and relaxationby 30% and 34.5%, respectively. Fractional shortening was reduced by26.1%, compared to Ad.I-1 WT (FIG. 2B). These results indicate thatphosphorylation of either Ser-67 or Thr-75 on I-1 exerts similardecrease on myocyte contractility, resulting in no further depression ofthe contractile parameters, when both sites are simultaneouslyphosphorylated.

To assess whether stimulation of the cAMP-dependent kinase pathway iscapable of reversing the depressed function of myocytes expressingconstitutively phosphorylated I-1 mutants, adenoviruses infectedcardiomyocytes were treated with a range of forskolin concentrationsfrom 10 nM to 1 μM. Surprisingly, high doses of forskolin elicitedarrhythmias only in myocytes expressing the constitutivelyphosphorylation sites (Ser-67 and/or Thr-75) but not I-1 WT or GFP.Therefore, 0.1 μM was established as the highest concentration thatinduced stimulation of contractility without eliciting arrhythmias.Forskolin treatment of myocytes expressing Ad.GFP caused dramaticincreases in the velocities of contraction (38%) and relaxation (51%),as well as in fractional shortening (8.5%), compare to the basal levels(FIG. 15). The total number of hearts was as follows: 10 (Ad.GFP), 5(Ad.I-1WT), 5 (Ad.I-1(S67D)), 6 (Ad.I-1(T75D)), and 6(Ad.I-1(S67D/T75D)), with 15-20 myocytes/heart.

The increases in performance in cells expressing Ad.I-1WT were similarto the control cells (36.5%, 49% and 15.5% for the velocities ofcontraction and relaxation, respectively, and fractional shortening).Importantly, cardiac function of myocytes infected with Ad.I-1(S67D),Ad.I-1(T75D) or Ad.I-1(S67D/T75D) also improved upon drug treatment, butthe cardiac parameters did not reach the maximal effect observed eitherin the Ad.I-1WT or Ad.GFP (FIG. 15). Of note, the percent increases inthe rates of contraction upon forskolin for the constitutivelyphosphorylated I-1 infected myocytes, approximated the percent increasesfound for the Ad.GFP and Ad.I-1WT infections, indicating no alterationsin the PKA-signaling pathway. Thus, although cardiac contractility ofmyocytes expressing the phosphorylated I-1 mutants can be improved bysimilar increments as I-1WT and GFP infected cells upon forskolintreatment, the overall function remains depressed in comparison to thecontrol cells.

Example 7 Sarcoplasmic Reticulum Ca²⁺ Uptake in Adenoviral-InfectedCardiomyocytes

To determine whether the depressed contractility associated withphosphorylation of I-1 at Thr-75 corresponded to alterations in thesarcoplasmic reticulum calcium transport function, the initial rates ofCa²⁺ transport were assessed over a wide range of [Ca²⁺], similar tothose present in vivo during relaxation and contraction. The reactionconditions were as follows: 37° C. using 5 mM ATP in 40 mM Imidazole(pH=7.0), 95 mM KCl, 5 mM NaN₃, 5 mM MgCl₂, 0.5 mM EGTA, and 5 mMK₂C₂O₄. The apparent affinity of the transport system for Ca²⁺ decreasedsignificantly in myocytes infected with the Ad.I-1 (T75D) (EC₅₀value=0.67±0.01 μM; n=3; ***p<0.001), compared to either Ad.I-1WT(0.33±0.01 μM; n=3) or Ad.GFP (0.28±0.006 μM; n=3) (FIG. 16A). However,the V_(max) of Ca²⁺ uptake was similar among all the three groups.Furthermore, there were no differences in the sarcoplasmic calcium pump(SERCA2a) and phospholamban (PLN) protein levels in these groups (FIG.16B). These data indicate that phosphorylation of I-1 by PKC-α atThr-75, may elicit depressed cardiac contractility by reducing the Ca²⁺affinity of SERCA2a.

The basal SR Ca²⁺ uptake rates were assessed in homogenates generatedfrom myocytes infected with adenoviruses expressing GFP, as a control,and the I-1 species: I-1WT, I-1(S67D), I-1 (T75D) or I-1(S67D/T75D),under conditions which restrict Ca-uptake to SR. Consistent with arecent study [10], infection with an adenovirus expressing GFP orwild-type I-1 exhibited similar SERCA EC₅₀ values (0.294±0.001 μM and0.336±0.013 μM, respectively) (FIGS. 17A and 17C). However, the apparentaffinity of SERCA for calcium was significantly lower in myocytesexpressing the I-1 (S67D), I-1(T75D) and I-1(S67D/T75D) mutants(0.457±0.012 μM, 0.664±0.014 μM and 0.611±0.005 μM, respectively) (FIGS.17A and 17C). The data shown in FIG. 17 were normalized to thecalculated V_(max) for each group, and fit to a sigmoidal curve by usingOriginLab 5.1 program.

It has, thus, been shown that phosphorylation of Ser-67 and/or Thr-75 onthe human I-1 isoform mitigates the effects of subsequent PKAstimulation in cardiomyocytes. Phosphorylation of either or both sitessimultaneously decrease the ability of I-1 to fully inhibit PP1 activityfollowing PKA activation, resulting in an overall impaired SERCA 2atransport function and cardiac contractility.

As expected, PKA stimulation by forskolin was associated with asignificant decrease in the SR Ca⁺²-uptake EC₅₀ for Ca⁺² in myocytesexpressing GFP (EC₅₀=0.17±0.029 μM), and this decrease was similar tothat observed in cardiomyocytes expressing wild-type I-1(EC₅₀=0.147±0.005 μM). (FIGS. 17B and 17C). However, although forskolintreatment of cardiomyocytes expressing the constitutively phosphorylatedI-1 mutants was capable of improving EC₅₀ values from their respectivebasal values, the calcium uptake rates remained depressed compare to theI-1 WT and GFP infected cells. The EC₅₀ values for cardiomyocytesexpressing I-1 (S67D), I-1(T75D) and I-1(S67D/T75D) following forskolintreatment were: 0.234±0.005 μM, 0.342±0.016 μM, and 0.334±0.053 μM,respectively (FIGS. 17A and 17C).

Thus, the consequences of constitutive phosphorylation of I-1 at Ser-67and/or Thr-75 were associated with depressed SR calcium uptake rates andcardiomyocyte function. Depressed SR calcium uptake rates werereflective of significantly higher SERCA EC₅₀ values under both basaland forskolin stimulated conditions. Although PKA activation byForskolin did not improve the Ca²⁺-ATPase function in myocytesexpressing phosphorylated Ser-67 and/or Thr-75 I-1 to the same extentthan myocytes expressing I-1WT or GFP, the percentage decreases of theEC₅₀ values appeared similar in all the groups, indicating that thePKA-signaling pathway is not altered.

Since calcium uptake into the SR represents a nodal point at whichcardiomyocyte contractility is regulated, the mechanical performance ofthe cardiomyocytes infected with phosphorylated Ser-67 and/or Thr75 I-1mirrored SR calcium uptake measurements. Phosphorylation of I-1 ateither Ser-67 and/or Thr-75 elicited significant depression of SERCAtransport function, and these values remained depressed even afterforskolin treatment. Indeed, stimulation of PKA in these groups ofinfected myocytes only improved SR Ca⁺² uptake values to the basallevels of the I-1WT group. These findings indicate that the effects ofPKA are blunted in cardiomyocytes by the constitutively phosphorylatedI-1 mutants at either Ser-67 or Thr-75. Similar values for the maximumvelocity (V_(max)) of Ca⁺² uptake were obtained in all samples.

Example 8 Protein Phosphatase Activity in Infected Cardiomyocytes

The decreases in cardiomyocyte contractility and the apparent affinityof the SR Ca²⁺-transport system for Ca²⁺ prompted investigation of theprotein phosphatase activity levels in adult rat cardiomyocytes infectedwith Ad.I-1WT, Ad.I-1 (T75D), and Ad.GFP. Total phosphatase activity wasassayed in cardiomyocyte lysates (1 μg) infected with Ad.GFP, Ad.I-1 WT,or Ad.I-1(T75D). The reaction mixture contained 50 mM Tris-HCl (pH 7.0),0.1 mM Na₂EDTA, 5 mM DTT, 0.01% Brij35, and radiolabeled Myelin BasicProtein (50 μM). Total phosphatase activity was increased by 16% inmyocytes expressing I-1(T75D), compared to cells expressing I-1wild-type or control cells (FIG. 18A). The relative contribution of thetwo major classes of cellular protein phosphatases, PP1 and PP2A, wasdetermined in the presence of okadaic acid at a concentration (10 nM)which inhibits PP2A more potently (Neumann et al. J. Mol. Cell. Cardiol(1997)). Cardiomyocytes infected with Ad.I-1 (T75D) showed a 27%increase in type-1 phosphatase activity compared to Ad.I-1WT and Ad.GFP,whereas there was no change in the type-2A phosphatase activity (FIG.18A). As shown in FIG. 13B, the protein levels of PP1 were the same inall cases.

The effects of I-1 phosphorylation at Thr-75 on type-I phosphataseactivity were further confirmed by measuring the activity of thepurified PP1 catalytic subunit (PP1c) in the presence of recombinant I-1wild-type, or I-1 with either constitutively phosphorylated I-1,I-1(T75D), or I-1(S67A) pre-phosphorylated by PKC-α. Both mutant I-1proteins significantly increased PP1c activity by 23% and 25%,respectively, compared to I-1 wild-type (FIG. 18B). Taken together,these data demonstrate that phosphorylation of I-1 at Thr-75 by PKC-αincreases PP1 activity in both isolated myocytes and in vitro systems.

After PKA stimulation by forskolin, although the percentage of totalprotein phosphatase inhibition appeared to be similar in all the groups(FIG. 19), selective PP1 inhibition, as assessed by using 10 nM Okadaicacid as a PP2A inhibitor (Rodriguez et al. J. Biol. Chem. (2006) Neumannet al. J. Mol Cell Cardiol. (1997)), by myocytes expressing Ad.I-1WT wassignificant higher upon forskolin treatment compared to cells infectedwith Ad.GFP. Okadaic acid (10 nM) was added to cell lysates todifferentiate type 1 and 2A phosphatase activities. The results indicatethat the levels of endogenous I-1 may not be sufficiently high to fullyinhibit PP1 activity. In contrast, the three constitutivelyphosphorylated I-1 mutants, I-1 (S67D), I-1 (T75D) and I-1 (S67D/T75D),presented significantly less inhibition of PP1 under forskolin, comparedto either control or wild-type infected cells. Thus, constitutivephosphorylation of either Ser-67 or Thr-75 reduced the extent to whichPP1 became inhibited, following PKA stimulation in cardiomyocytes.

These two sites appeared equivalent in eliciting this effect, and noadditive effect was observed when both sites were phosphorylated.Indeed, constitutive phosphorylation of both or either of these sitescaused PP1 activity to remain-2-fold higher following PKA stimulation.These data indicate that, in the failing heart, attenuated β-adrenergicsignaling and increased PKC signaling would serve as a double insult,favoring higher PP1 activity. The percentage of PP1 inhibition inmyocytes expressing the I-1 double mutant (S67D/T75D) was similar tothat exhibited by either S67D or T75D I-1 mutants, indicating thatsimultaneous phosphorylation of the two sites did not exhibit anadditive effect in the inhibition of PP1 activity after PKA stimulation.

Example 9 Effect of Phosphorylation of I-1 at Ser-67 and Thr-75 upon itsAbility to be a Substrate for PKA

To analyze whether phosphorylation of I-1 at both Ser-67 and Thr-75reduces the ability of PKA phosphorylation of I-1 at Thr-35, recombinantI-1 wild-type and I-1(S67D/T75D) proteins were incubated in vitro withPKA in the presence of [γ-³²P] ATP. As shown in FIG. 20, the doublemutant incorporated 29% less radioactivity than the wild-type,indicating that Thr-35 can not be phosphorylated to the same extent inthe mutant. Thus, the ability of I-1 to be phosphorylated by PKA islikely altered when both Ser-67 and Thr-75 are previouslyphosphorylated.

Taken together, the data described herein indicate that, in the heart,phosphorylation of Ser-67 and/or Thr-75 on the human I-1 isoform maywork to partially suppress β-adrenergic signaling and, consequently,reduce the stimulatory effects on contractility through the maintenanceof an abnormally enhance PP1 activity.

Example 10 Codon-optimized Truncated I-1

The wild-type I-1 gene was found to use rare codons with a highfrequency and to contain several negatively cis-acting motifs, whichmight hamper expression in animals. Thus, standard codon optimization(based on the human codon usage table, below, as published onhttp://bip.weizmann.ac.il/index.html and described, for example, in“Genetic Databases”, M. J. Bishop ed., Academic Press, (1999) wasemployed to synthesize a truncated mutant human I-1 protein (T35D)encoded by SEQ ID NO: 13, below. The truncation of the I-1 cDNA is toencode the first 65 amino acids of the protein.

The codon usage was adapted to the codon bias of Homo sapiens genes,resulting in a high codon adaptation index (CAI) value (0.99). Thecodon-optimized (truncated) protein has an increased GC content(relative to the native human sequence) for more efficient AAV packaging(data not shown). Certain cis-acting sequence motifs were avoided (forexample, splice sites, polyA signals). Kozak sequence was introduced toincrease translational initiation. Two STOP codons were added to ensureefficient termination.

THE Human Codon Usage TABLE Gly GGG 17.08 0.23 Arg AGG 12.09 0.22 TrpTGG 14.74 1.00 Arg CGG 10.40 0.19 Gly GGA 19.31 0.26 Arg AGA 11.73 0.21End TGA 2.64 0.61 Arg CGA 5.63 0.10 Gly GGT 13.66 0.18 Ser AGT 10.180.14 Cys TGT 9.99 0.42 Arg CGT 5.16 0.09 Gly GGC 24.94 0.33 Ser AGC18.54 0.25 Cys TGC 13.86 0.58 Arg CGC 10.82 0.19 Glu GAG 38.82 0.59 LysAAG 33.79 0.60 End TAG 0.73 0.17 Gln CAG 32.95 0.73 Glu GAA 27.51 0.41Lys AAA 22.32 0.40 End TAA 0.95 0.22 Gln CAA 11.94 0.27 Asp GAT 21.450.44 Asn AAT 16.43 0.44 Tyr TAT 11.80 0.42 His CAT 9.56 0.41 Asp GAC27.06 0.56 Asn AAC 21.30 0.56 Tyr TAC 16.48 0.58 His CAC 14.00 0.59 ValGTG 28.60 0.48 Met ATG 21.86 1.00 Leu TTG 11.43 0.12 Leu CTG 39.93 0.43Val GTA 6.09 0.10 Ile ATA 6.05 0.14 Leu TTA 5.55 0.06 Leu CTA 6.42 0.07Val GTT 10.30 0.17 Ile ATT 15.03 0.35 Phe TTT 15.36 0.43 Leu CTT 11.240.12 Val GTC 15.01 0.25 Ile ATC 22.47 0.52 Phe TTC 20.72 0.57 Leu CTC19.14 0.20 Ala GCG 7.27 0.10 Thr ACG 6.80 0.12 Ser TCG 4.38 0.06 Pro CCG7.02 0.11 Ala GCA 15.50 0.22 Thr ACA 15.04 0.27 Ser TCA 10.96 0.15 ProCCA 17.11 0.27 Ala GCT 20.23 0.28 Thr ACT 13.24 0.23 Ser TCT 13.51 0.18Pro CCT 18.03 0.29 Ala GCC 28.43 0.40 Thr ACC 21.52 0.38 Ser TCC 17.370.23 Pro CCC 20.51 0.33For each codon, the table displays the frequency of usage of each codon(per thousand) in human coding regions (first column) and the relativefrequency of each codon among synonymous codons (second column).SEQ ID NO: 13 reads as follows:

GGGCGAATTGGGTACCGCCACCATGGAACAGGACAACAGCCCCCGGAAGATCCAGTTCACCGTGCCCCTGCTGGAACCCCACCTGGACCCCGAGGCCGCCGAGCAGATCCGGCGGAGAAGGCCCGACCCCGCCACCCTGGTGCTGACCAGCGACCAGAGCAGCCCCGAGATCGACGAGGACCGGATCCCCAACCCCCACCTGAAGAGCACCCTGGCCTGATGAGAGCTCCAGCTTTTGTTCCCThe amino acid sequence encoded by SEQ ID NO: 13 is set forth as SEQ IDNO: 14, as follows:

MEQDNSPRKIQFTVPLLEPHLDPEAAEQIRRRRPDPATLVLTSDQSSPEIDEDRIPNPHLKSTLAThe amino acid sequence encoded by SEQ ID NO: 13 is set forth as SEQ IDNO: 14.

Codon optimization for I-1 mutants described herein is likewisecontemplated.

Sequence Information

It should be understood that for purposes of describing, defining, andclaiming the present invention, reference to a “SEQ ID NO:” is taken toinclude all sequences having at least 90 percent identity therewith andretaining any specified mutation. In more specific embodiments, it isunderstood to include sequences having at least 95% identity therewithand retaining any specified mutation. In yet more specific embodiments,it includes sequences having between 99% and 100% identity therewith andretaining any specified mutation.

The wild type sequence for Homo Sapiens Protein Phosphatase I, inhibitorsubunit 1A, (PP1I1A) mRNA, is set forth below (SEQ ID NO:7). Nucleotidechanges for mutants occur between positions 361 and 400, according tothis numbering scheme based on the cDNA. The first A in the codingsequence could be indicated as 1 (rather than 172).

  1 AGTGTCCCCG GAGCCGCGAG CTGGGAGCGC TGTGCCGGGA GCCGGGAGCC GAGCGCGCCG 61 GGCTGGGGCC CGCGCCGGAG CGGAGCGGAG AGGGAGCGCG CCCGCCCCAG CCCCGAGTCC121 CGCCGCCTTC CCTCCCGCCG CAGCGCGGGC CCACCGGCCG CCGCCCCAGC CATGGAGCAA181 GACAACAGCC CCCAAAAGAT CCAGTTCACG GTCCCGCTGC TGGAGCCGCA CCTTGACCCC241 GAGGCGGCGG AGCAGATTCG GAGGCGCCGC CCC ACC CCTG CCACCCTCGT GCTGACCAGT301 GACCAGTCAT CCCCAGAGAT AGATGAAGAC CGGATCCCCA ACCCACATCT CAAGTCCACT361 TTGGCAATG T CT CCACGGCA ACGGAAGAAG ATG ACAAGGA TCACACCCAC AATGAAAGAG 421CTCCAGATGA TCGTTGAACA TCACCTGGGG CAACAGCAGC AAGGAGAGGA ACCTCAGGGG 481GCCGCTGAGA GCACAGGAAC CCAGGAGTCC CGCCCACCTG GGATCCCAGA CACAGAAGTG 541GAGTCAAGGC TGGGCACCTC TGGGACAGCA AAAAAAACTG CAGAATGCAT CCCTAAAACT 601CACGAAAGAG GCAGTAAGGA ACCCAGCACA AAAGAACCCT CAACCCATAT ACCACCACTG 661GATTCCAAGG GAGCCAACTC GGTCTGAGAG AGGAGGAGGT ATCTTGGGAT CAAGACTGCA

The amino acid sequence encoded by SEQ ID NO: 7 is set forth as SEQ IDNO: 8, as follows:

  1 MEQDNSPQKIQFTVPLLEPHLDPEAAEQIRRRRP T PATLVLTSDQSSPEI  51DEDRIPNPHLKSTLAMSPRQRKKM T RITPTMKELQMMVEHHLGQQQ 101QGEEPEGAAESTGTQESRPPGIPDTEVESRLGTSGTAKKTAECIPKTHER 151GSKEPSTKEPSTHIPPLDSKGANSVAmino acid positions 35, 67, and 75 have been highlighted with bold fontand underlining.

It is understood that the human wild type PP-1 exists as polymorphs,known and possibly unknown. For example, there are two polymorphs basedon amino acid position 8, which may comprise either an asparagine orlysine residue. The former is known in the art as the Q variant, whilethe latter is known in the art as the K variant. As mentioned in the“Definitions” section, above, it will be apparent to one of ordinaryskill in the art that either variant is suitable, and the variants areessentially interchangeable, for purposes of practicing and defining thepresent invention. It will be equally apparent that other suchpolymorphs may exist and are equally intended to fall within the scopeof the present invention.

It should be noted that nucleotide position 172 of SEQ ID NO: 7, above,equals position 1, for the purposes of describing the sequences herein.The relevant coding sequence ends at position 688. The location of thecodon corresponding to amino acid position 67 of the polypeptide encodedby the nucleotide sequence defined by positions 172-688, above,according to the present invention, is: positions 371, 372, 373, (TCT),and the location for the codon corresponding to amino acid position 75,similarly, is 394, 395, 396 (ACA).

The mutant “I-1 S67A” has the codon TCT replaced by GCA and is set forthas SEQ ID NO: 1. The mutant “I-1 S67D” has the codon TCT replaced by GACand is set forth as SEQ ID NO: 2. The mutant “I-1 T75D” has the codonACA replaced by GAC and is set forth as SEQ ID NO: 3. The mutant “I-1T75A” has the codon ACA replaced by GCA and is set forth as SEQ ID NO:4.

The amino acid sequence encoded by SEQ ID NO: 3 is set forth as SEQ IDNO: 5. The amino acid sequence encoded by SEQ ID NO: 4 is set forth asSEQ ID NO: 6. The amino acid sequence encoded by SEQ ID NO: 7 is setforth as SEQ ID NO: 8.

The mutant “I-1 S67C” has the codon TCT replaced by the codon TGT or TGCand is set forth as SEQ ID NO: 9. The mutant “I-1 T75C” has the codonACA replaced by the codon TGT or TGC and is set forth as SEQ ID NO: 10.The amino acid sequence encoded by SEQ ID NO: 9 is set forth as SEQ IDNO:11. The amino acid sequence encoded by SEQ ID NO: 10 is set forth asSEQ ID NO: 12.

It is also contemplated that a single nucleic acid molecule may comprisemutations at both locations. The mutant “I-1 S67A/T75A” has codon 67(TCT) replaced by GCA and codon 75 (ACA) replaced by GCA and is setforth in SEQ ID NO: 15. The amino acid sequence encoded by SEQ ID NO: 15is set forth as SEQ ID NO: 16. The mutant “I-1 S67D/T75D” has codon 67(TCT) replaced by GAC and codon 75 (ACA) replaced by GAC and is setforth in SEQ ID NO: 17. The amino acid sequence encoded by SEQ ID NO: 17is set forth as SEQ ID NO: 18.

The mutant “I-1 T35D” has the codon ACC replaced by the codon GAC and isset forth in SEQ ID NO: 19.

The amino acid sequence encoded by SEQ ID NO: 19 is set forth as SEQ IDNO: 20. The mutant “I-1 S67A/T35D” has the codon TCT (371-373) replacedby GCA and the codon ACC (277-279) replaced by GAC and is set forth inSEQ ID NO: 22. The amino acid sequence encoded by SEQ ID NO: 22 is setforth as SEQ ID NO: 21.

SEQ ID NO: 1:                                                        ATGGAGCAA 181GACAACAGCC CCCAAAAGAT CCAGTTCACG GTCCCGCTGC TGGAGCCGCA CCTTGACCCC 241GAGGCGGCGG AGCAGATTCG GAGGCGCCGC CCCACCCCTG CCACCCTCGT GCTGACCAGT 301GACCAGTCAT CCCCAGAGAT AGATGAAGAC CGGATCCCCA ACCCACATCT CAAGTCCACT 361TTGGCAATGG CACCACGGCA ACGGAAGAAG ATGACAAGGA TCACACCCAC AATGAAAGAG 421CTCCAGATGA TGGTTGAACA TCACCTGGGG CAACAGCAGC AAGGAGAGGA ACCTGAGGGG 481GCCGCTGAGA GCACAGGAAC CCAGGAGTCC CGCCCACCTG GGATCCCAGA CACAGAAGTG 541GAGTCAAGGC TGGGCACCTC TGGGACAGCA AAAAAAACTG CAGAATGCAT CCCTAAAACT 601CACGAAAGAG GCAGTAAGGA ACCCAGCACA AAAGAACCCT CAACCCATAT ACCACCACTG 661GATTCCAAGG GAGCCAACTC GGTCTGA SEQ ID NO: 2:                                                        ATGGAGCAA 181GACAACAGCC CCCAAAAGAT CCAGTTCACG GTCCCGCTGC TGGAGCCGCA CCTTGACCCC 241GAGGCGGCGG AGCAGATTCG GAGGCGCCGC CCCACCCCTG CCACCCTCGT GCTGACCAGT 301GACCAGTCAT CCCCAGAGAT AGATGAAGAC CGGATCCCCA ACCCACATCT CAAGTCCACT 361TTGGCAATGG ACCCACGGCA ACGGAAGAAG ATGACAAGGA TCACACCCAC AATGAAAGAG 421CTCCAGATGA TGGTTGAACA TCACCTGGGG CAACAGCAGC AAGGAGAGGA ACCTGAGGGG 481GCCGCTGAGA GCACAGGAAC CCAGGAGTCC CGCCCACCTG GGATCCCAGA CACAGAAGTG 541GAGTCAAGGC TGGGCACCTC TGGGACAGCA AAAAAAACTG CAGAATGCAT CCCTAAAACT 601CACGAAAGAG GCAGTAAGGA ACCCAGCACA AAAGAACCCT CAACCCATAT ACCACCACTG 661GATTCCAAGG GAGCCAACTC GGTCTGA SEQ ID NO: 3:                                                        ATGGAGCAA 181GACAACAGCC CCCAAAAGAT CCAGTTCACG GTCCCGCTGC TGGAGCCGCA CCTTGACCCC 241GAGGCGGCGG AGCAGATTCG GAGGCGCCGC CCCACCCCTG CCACCCTCGT GCTGACCAGT 301GACCAGTCAT CCCCAGAGAT AGATGAAGAC CGGATCCCCA ACCCACATCT CAAGTCCACT 361TTGGCAATGT CTCCACGGCA ACGGAAGAAG ATGGACAGGA TCACACCCAC AATGAAAGAG 421CTCCAGATGA TGGTTGAACA TCACCTGGGG CAACAGCAGC AAGGAGAGGA ACCTGAGGGG 481GCCGCTGAGA GCACAGGAAC CCAGGAGTCC CGCCCACCTG GGATCCCAGA CACAGAAGTG 541GAGTCAAGGC TGGGCACCTC TGGGACAGCA AAAAAAACTG CAGAATGCAT CCCTAAAACT 601CACGAAAGAG GCAGTAAGGA ACCCAGCACA AAAGAACCCT CAACCCATAT ACCACCACTG 661GATTCCAAGG GAGCCAACTC GGTCTGA SEQ ID NO: 4:                                                        ATGGAGCAA 181GACAACAGCC CCCAAAAGAT CCAGTTCACG GTCCCGCTGC TGGAGCCGCA CCTTGACCCC 241GAGGCGGCGG AGCAGATTCG GAGGCGCCGC CCCACCCCTG CCACCCTCGT GCTGACCAGT 301GACCAGTCAT CCCCAGAGAT AGATGAAGAC CGGATCCCCA ACCCACATCT CAAGTCCACT 361TTGGCAATGT CTCCACGGCA ACGGAAGAAG ATGGCAAGGA TCACACCCAC AATGAAAGAG 421CTCCAGATGA TGGTTGAACA TCACCTGGGG CAACAGCAGC AAGGAGAGGA ACCTGAGGGG 481GCCGCTGAGA GCACAGGAAC CCAGGAGTCC CGCCCACCTG GGATCCCAGA CACAGAAGTG 541GAGTCAAGGC TGGGCACCTC TGGGACAGCA AAAAAAACTG CAGAATGCAT CCCTAAAACT 601CACGAAAGAG GCAGTAAGGA ACCCAGCACA AAAGAACCCT CAACCCATAT ACCACCACTG 661GATTCCAAGG GAGCCAACTC GGTCTGA SEQ ID NO: 5:   1MEQDNSPQKIQFTVPLLEPHLDPEAAEQIRRRRPTPATLVLTSDQSSPEI  51DEDRIPNPHLKSTLAMSPRQRKKM D RITPTMKELQMMVEHHLGQQQ 101QGEEPEGAAESTGTQESRPPGIPDTEVESRLGTSGTAKKTAECIPKTHER 151GSKEPSTKEPSTHIPPLDSKGANSV SEQ ID NO: 6:   1MEQDNSPQKIQFTVPLLEPHLDPEAAEQIRRRRPTPATLVLTSDQSSPEI  51DEDRIPNPHLKSTLAMSPRQRKKM A RITPTMKELQMMVEHHLGQQQ 101QGEEPEGAAESTGTQESRPPGIPDTEVESRLGTSGTAKKTAECIPKTHER 151GSKEPSTKEPSTHIPPLDSKGANSV SEQ ID NO: 9:                                                        ATGGAGCAA 181GACAACAGCC CCCAAAAGAT CCAGTTCACG GTCCCGCTGC TGGAGCCGCA CCTTGACCCC 241GAGGCGGCGG AGCAGATTCG GAGGCGCCGC CCCACCCCTG CCACCCTCGT GCTGACCAGT 301GACCAGTCAT CCCCAGAGAT AGATGAAGAC CGGATCCCCA ACCCACATCT CAAGTCCACT 361TTGGCAATGT G(T/C)CCACGGCA ACGGAAGAAG ATGACAAGGA TCACACCCAC AATGAAAGAG421 CTCCAGATGA TGGTTGAACA TCACCTGGGG CAACAGCAGC AAGGAGAGGA ACCTGAGGGG481 GCCGCTGAGA GCACAGGAAC CCAGGAGTCC CGCCCACCTG GGATCCCAGA CACAGAAGTG541 GAGTCAAGGC TGGGCACCTC TGGGACAGCA AAAAAAACTG CAGAATGCAT CCCTAAAACT601 CACGAAAGAG GCAGTAAGGA ACCCAGCACA AAAGAACCCT CAACCCATAT ACCACCACTG661 GATTCCAAGG GAGCCAACTC GGTCTGA SEQ ID NO: 10:                                                        ATGGAGCAA 181GACAACAGCC CCCAAAAGAT CCAGTTCACG GTCCCGCTGC TGGAGCCGCA CCTTGACCCC 241GAGGCGGCGG AGCAGATTCG GAGGCGCCGC CCCACCCCTG CCACCCTCGT GCTGACCAGT 301GACCAGTCAT CCCCAGAGAT AGATGAAGAC CGGATCCCCA ACCCACATCT CAAGTCCACT 361TTGGCAATGT CTCCACGGCA ACGGAAGAAG ATGTG(T/C)AGGA TCACACCCAC AATGAAAGAG421 CTCCAGATGA TGGTTGAACA TCACCTGGGG CAACAGCAGC AAGGAGAGGA ACCTGAGGGG481 GCCGCTGAGA GCACAGGAAC CCAGGAGTCC CGCCCACCTG GGATCCCAGA CACAGAAGTG541 GAGTCAAGGC TGGGCACCTC TGGGACAGCA AAAAAAACTG CAGAATGCAT CCCTAAAACT601 CACGAAAGAG GCAGTAAGGA ACCCAGCACA AAAGAACCCT CAACCCATAT ACCACCACTG661 GATTCCAAGG GAGCCAACTC GGTCTGA SEQ ID NO: 11:   1MEQDNSPQKIQFTVPLLEPHLDPEAAEQIRRRRPTPATLVLTSDQSSPEI  51 DEDRIPNPHLKSTLAMC PRQRKKMTRITPTMKELQMMVEHHLGQQQ 101QGEEPEGAAESTGTQESRPPGIPDTEVESRLGTSGTAKKTAECIPKTHER 151GSKEPSTKEPSTHIPPLDSKGANSV SEQ ID NO: 12:   1MEQDNSPQKIQFTVPLLEPHLDPEAAEQIRRRRPTPATLVLTSDQSSPEI  51DEDRIPNPHLKSTLAMSPRQRKKM C RITPTMKELQMMVEHHLGQQQ 101QGEEPEGAAESTGTQESRPPGIPDTEVESRLGTSGTAKKTAECIPKTHER 151GSKEPSTKEPSTHIPPLDSKGANSV SEQ ID NO: 15:                                                        ATGGAGCAA 181GACAACAGCC CCCAAAAGAT CCAGTTCACG GTCCCGCTGC TGGAGCCGCA CCTTGACCCC 241GAGGCGGCGG AGCAGATTCG GAGGCGCCGC CCCACCCCTG CCACCCTCGT GCTGACCAGT 301GACCAGTCAT CCCCAGAGAT AGATGAAGAC CGGATCCCCA ACCCACATCT CAAGTCCACT 361TTGGCAATGG CACCACGGCA ACGGAAGAAG ATGGCAAGGA TCACACCCAC AATGAAAGAG 421CTCCAGATGA TGGTTGAACA TCACCTGGGG CAACAGCAGC AAGGAGAGGA ACCTGAGGGG 481GCCGCTGAGA GCACAGGAAC CCAGGAGTCC CGCCCACCTG GGATCCCAGA CACAGAAGTG 541GAGTCAAGGC TGGGCACCTC TGGGACAGCA AAAAAAACTG CAGAATGCAT CCCTAAAACT 601CACGAAAGAG GCAGTAAGGA ACCCAGCACA AAAGAACCCT CAACCCATAT ACCACCACTG 661GATTCCAAGG GAGCCAACTC GGTCTGA SEQ ID NO: 16:   1MEQDNSPQKIQFTVPLLEPHLDPEAAEQIRRRRPTPATLVLTSDQSSPEI  51 DEDRIPNPHLKSTLAMA PRQRKKM A RITPTMKELQMMVEHHLGQQQ 101QGEEPEGAAESTGTQESRPPGIPDTEVESRLGTSGTAKKTAECIPKTHER 151GSKEPSTKEPSTHIPPLDSKGANSV SEQ ID NO: 17:                                                        ATGGAGCAA 181GACAACAGCC CCCAAAAGAT CCAGTTCACG GTCCCGCTGC TGGAGCCGCA CCTTGACCCC 241GAGGCGGCGG AGCAGATTCG GAGGCGCCGC CCCACCCCTG CCACCCTCGT GCTGACCAGT 301GACCAGTCAT CCCCAGAGAT AGATGAAGAC CGGATCCCCA ACCCACATCT CAAGTCCACT 361TTGGCAATGG ACCCACGGCA ACGGAAGAAG ATGGACAGGA TCACACCCAC AATGAAAGAG 421CTCCAGATGA TGGTTGAACA TCACCTGGGG CAACAGCAGC AAGGAGAGGA ACCTGAGGGG 481GCCGCTGAGA GCACAGGAAC CCAGGAGTCC CGCCCACCTG GGATCCCAGA CACAGAAGTG 541GAGTCAAGGC TGGGCACCTC TGGGACAGCA AAAAAAACTG CAGAATGCAT CCCTAAAACT 601CACGAAAGAG GCAGTAAGGA ACCCAGCACA AAAGAACCCT CAACCCATAT ACCACCACTG 661GATTCCAAGG GAGCCAACTC GGTCTGA SEQ ID NO: 18:   1MEQDNSPQKIQFTVPLLEPHLDPEAAEQIRRRRPTPATLVLTSDQSSPEI  51 DEDRIPNPHLKSTLAMD PRQRKKM D RITPTMKELQMMVEHHLGQQQ 101QGEEPEGAAESTGTQESRPPGIPDTEVESRLGTSGTAKKTAECIPKTHER 151GSKEPSTKEPSTHIPPLDSKGANSV SEQ ID NO: 19:                                                        ATGGAGCAA 181GACAACAGCC CCCAAAAGAT CCAGTTCACG GTCCCGCTGC TGGAGCCGCA CCTTGACCCC 241GAGGCGGCGG AGCAGATTCG GAGGCGCCGC CCCGACCCTG CCACCCTCGT GCTGACCAGT 301GACCAGTCAT CCCCAGAGAT AGATGAAGAC CGGATCCCCA ACCCACATCT CAAGTCCACT 361TTGGCA SEQ ID NO: 20:   1 MEQDNSPQKIQFTVPLLEPHLDPEAAEQIRRRRP DPATLVLTSDQSSPEI  51 DEDRIPNPHLKSTLA SEQ ID NO: 21:   1MEQDNSPQKIQFTVPLLEPHLDPEAAEQIRRRRP D PATLVLTSDQSSPEI  51DEDRIPNPHLKSTLAM A PRQRKKMTRITPTMKELQMMVEHHLGQQQ 101QGEEPEGAAESTGTQESRPPGIPDTEVESRLGTSGTAKKTAECIPKTHER 151GSKEPSTKEPSTHIPPLDSKGANSV SEQ ID NO: 22:                                                        ATGGAGCAA 181GACAACAGCC CCCAAAAGAT CCAGTTCACG GTCCCGCTGC TGGAGCCGCA CCTTGACCCC 241GAGGCGGCGG AGCAGATTCG GAGGCGCCGC CCCGACCCTG CCACCCTCGT GCTGACCAGT 301GACCAGTCAT CCCCAGAGAT AGATGAAGAC CGGATCCCCA ACCCACATCT CAAGTCCACT 361TTGGCAATGG CACCACGGCA ACGGAAGAAG ATGACAAGGA TCACACCCAC AATGAAAGAG 421CTCCAGATGA TGGTTGAACA TCACCTGGGG CAACAGCAGC AAGGAGAGGA ACCTGAGGGG 481GCCGCTGAGA GCACAGGAAC CCAGGAGTCC CGCCCACCTG GGATCCCAGA CACAGAAGTG 541GAGTCAAGGC TGGGCACCTC TGGGACAGCA AAAAAAACTG CAGAATGCAT CCCTAAAACT 601CACGAAAGAG GCAGTAAGGA ACCCAGCACA AAAGAACCCT CAACCCATAT ACCACCACTG 661GATTCCAAGG GAGCCAACTC GGTCTGA

1. An isolated nucleic acid molecule that encodes a variant phosphataseinhibitor-1 protein, wherein the nucleic acid molecule encodes apolypeptide having at least 90% identity to: (a) SEQ ID NO:6 or afragment of SEQ ID NO: 6; (b) SEQ ID NO: 21 or a fragment of SEQ ID NO:21; or (c) SEQ ID NO:16 or a fragment of SEQ ID NO: 16, and wherein thepolypeptide or the fragment comprises: (i) a constitutivelyphosphorylated amino acid at position 35, said constitutivelyphosphorylated amino acid being aspartic acid or glutamic acid and (ii)a constitutively unphosphorylated amino acid at positions 67 and/or 75,wherein the polypeptide or fragment inhibits the activity of a proteinphosphatase I as determined by an assay using a rabbit proteinphosphatase I.
 2. The isolated nucleic acid molecule of claim 1, whichis selected from the group consisting of a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 4, a nucleotidemolecule which is at least 90% identical to the nucleotide sequence ofSEQ ID NO: 4, the nucleotide sequence of SEQ ID NO: 22, a nucleotidemolecule which is at least 90% identical to the nucleotide sequence ofSEQ ID NO: 22, and the nucleotide sequence of SEQ ID NO: 15, and anucleotide molecule which is at least 90% identical to the nucleotidesequence of SEQ ID NO:
 15. 3. A recombinant vector comprising theisolated nucleic acid molecule according to claim
 1. 4. A recombinanthost cell comprising the recombinant vector of claim
 3. 5. A method forproducing and isolating a protein, wherein the method encompassesculturing the host cell of claim 4 under conditions in which the proteinis expressed and isolating the expressed protein.